Method for Determining the Propensity for Calcification

ABSTRACT

The present invention relates to a method for determining the propensity of a fluid for calcification.

The present invention relates to a method for determining the propensityof a fluid for calcification.

In nature, the deposition of calcium salts is an abundant phenomenonfound in living and non-living environments, in vivo and in vitro. Itmay occur whenever fluids comprising calcium salts get in contact withsurfaces or may even occur in solution. A high degree of calcificationmay harm technical devices in vitro and may harm patients when it occursin vivo, in particular in the cardiovascular system and in soft tissues.

Today, cardiovascular and soft tissue calcifications are a major healthproblem and one of the leading causes of death worldwide. Calcificationis a widespread and highly important process characterized by thedeposition of calcium salts that may occur throughout the whole body.Typical examples for the precipitation of calcium salts are theprecipitation of calcium phosphates, calcium carbonates and complexesthereof.

Interestingly, in vivo, calcium and phosphate concentrations aretypically supersaturated in most tissues and body fluids throughout thebody, thereby creating a continuous chemical pressure towardscalcification. Under physiological conditions however, calcium andphosphate only precipitate in bones and teeth, whereas soft tissues in ahealthy patient do not or merely slightly and slowly calcify. Thisindicates that, in vivo, the precipitation of calcium is awell-regulated and site-specific process.

However, calcification may also occur in other tissues and organs andmay, hereby, be pathological. For instance, calcification may cause softtissues such as the skin, brown fat, lung, kidney, heart or a joint toharden. Here, calcification may lead to a pathological tissue andpotentially to a corresponding pathological condition including theclinical symptoms thereof. Moreover, calcification may occur in thelumen of certain organs such as the kidney, in particular the renalpelvis, the ureter, the bladder, the gallbladder and the bile duct.Here, a kidney stone (nephrolith) and a gallstone, respectively, mayoccur.

Nevertheless, the most common pathological calcification isatherosclerosis. Mild forms of atherosclerosis mostly occur in elderlypeople without provoking any clinical symptoms. However, cardiovascularcalcification may increase the risk of numerous diseases such as, e.g.,hypertonia, myocardial infarction, limb ischemia and cerebral apoplexy(stroke). These cardiovascular diseases have become the leading cause ofdeath and a major challenge for healthcare Systems worldwide (Yusuf etal., 2001). Highly pronounced forms of calcification may further lead tothe necessity of amputation of an extremity such as, e.g., a leg, anarm, a foot or a hand. The process of in vivo cardiovascular and softtissue calcification is not fully understood so far. It is consideredthat several chemical factors may play an important role incalcification. Such chemical factors may be inter alia vitamin D, oxalicacid, cortisol and calcium-binding polypeptides.

Cardiovascular and soft tissue calcification may be regarded as theresult of actively regulated cellular processes leading to atransformation of vascular smooth muscle cells into osteoblast-likecells (Reynolds et al, 2004; Reynolds et al, 2005). These cells areconsidered to process and handle surplus amounts of calcium andphosphate and deposit these extracellularly in the form of crystallinehydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) which is also the main component of theinorganic bone substance.

As an additional conceptual view on pathologic biomineralization, it isconsidered that the process of calcification also may be of a passiveand unregulated nature and that it takes place preferentially insituations characterized by an unfavorable interplay of serum-boundfactors of calcification (Jahnen-Dechent et al, 2001; Schafer et al.,2003; Heiss et al., 2008). Blood serum components may jointly undertakethe task of preventing the supersaturated calcium and phosphateconcentrations from precipitating. Calcium may be bound incalcium-comprising particles (Wald et al., 2011; Heiss et al., 2003),which may spontaneously convert into secondary calcium-comprisingparticles in a transitional ripening step associated with an increase inparticle diameter (Wald et al., 2011). Herein, fetuin-A is known toserve as a regulator of calcified matrix metabolism (Jahnen-Dechent etal., 2011).

Due to the severe consequences that calcification may have, it is ofconsiderable importance to have a reliable method for determining theoverall propensity of a body fluid for calcification, taking intoaccount all known and unknown factors inhibiting and promotingcalcification in the fluid. This is of particular importance fordiagnosing the risk of calcification in a patient who has developedcalcified plaques or is at risk of developing calcified plaques. Therisk of calcification is especially high in patients with reduced orabsent function of the kidney, the major organ of mineral metabolism.Thus dialysis patients have an exceptionally high risk of vascular andvalvular calcification with highly increased morbidity and mortality.

In the state of the art, the propensity for calcification in anindividual who is potentially at risk of developing calcified plaques isassessed by determining the phosphate concentration or the calciumphosphate product in the serum of the patient. If this concentrationexceeds a certain threshold, phosphate binders are administered in orderto decrease the phosphate uptake and thereby to prevent calciumprecipitation.

However, the phosphate concentration in the serum does not necessarilycorrelate with the propensity for calcification. In fact, there areindividuals bearing a normal phosphate concentration in the serum but,nevertheless, have a severely increased risk of developing calcifiedplaques or even have developed calcified plaques already. On the otherhand, there are also individuals who bear an increased phosphateconcentration in the serum, but who never develop any calcified plaques.

In the view of the above, the methods for determining the propensity ofa fluid for calcification known in the art are not reliable as they donot provide any information of the overall propensity of said fluid forcalcification.

Therefore, today, there is still an unmet need for reliable methods fordetermining the overall propensity of a fluid for calcification. This isof particular importance for diagnosing the risk of calcification in apatient who has developed calcified plaques or is at risk of developingcalcified plaques.

Surprisingly, we found that the overall propensity of a fluid forcalcification can be determined by a method based on determining therate of the formation of primary and/or secondary CPPs, the amount ofprimary and/or secondary CPPs and/or the rate of the transition ofprimary CPPs into secondary CPPs.

The present invention relates to a method for determining the propensityof a fluid for calcification, wherein said method is characterized bythe following steps:

-   (i) adding a soluble calcium salt and a soluble phosphate salt to a    sample of said fluid;-   (ii) incubating said sample at conditions allowing the formation of    calciprotein particles (CPPs); and-   (iii) determining one or more of the following:    -   (a) the rate of the formation of primary and/or secondary CPPs;    -   (b) the amount of primary and/or secondary CPPs; and/or    -   (c) the rate of the transition of primary CPPs into secondary        CPPs,        wherein an increase in one or more of (a), (b) and/or (c) of        step (iii) indicates an increased propensity of said fluid for        calcification.

As used herein, the term “calcification” may be understood in thebroadest sense as any deposition and/or precipitation of poorly solubleor insoluble calcium salts and/or the formation of calciproteinparticles (CPPs) in vivo and in vitro. As used herein, the terms“calcification”, “calcinations”, “calcium deposition” and “calciumprecipitation” may be understood interchangeably. Optionally,calcification may lead to the formation of calcified plaque(s), may bemicrocalcification and/or may be the precipitation of calcium salts insolution, thus, the formation of insoluble or poorly solublecalcium-comprising particles, including but not limited to primaryand/or secondary CPPs.

As used in the context of the present invention, the term “poorlysoluble” may be understood in the sense that the salt may bear asolubility of less than 10 mM, less than 1 mM, less than 0.1 mM, lessthan 10 less than 1 less than 0.1 μM or less than 10 nM in water at 20°C. The term “insoluble” may be understood in the sense that the salt maybear a solubility of less than 10 nM, less than 1 nM or less than 0.1 nMin water at 20° C.

Preferably, calcification is calcification in vivo. This means that thecalcification occurs in the fluid when the fluid is within a body of asubject and the propensity for said in vivo calcification is determinedby the present method. The fact that calcification may be calcificationin vivo may not exclude that the method may also be performed in vitro.As used throughout the invention, the terms “perform” and “conduct” maybe understood interchangeably. Preferably, the method itself isperformed in vitro, thus, is not practiced on the human or animal body.However, the results obtained by the method are usable for diagnosis ofthe patient.

In the context of calcification in vivo, calcification may also beunderstood as the formation of one or more calcified plaque(s). In thiscontext, the terms “calcified plaque” and “calcified lesion” may beunderstood interchangeably. Plaque formation may occur in various areasof the patient's body. More preferably, calcification is vascularcalcification, valvular calcification, cardiovascular calcification orcalcification of one or more soft tissue(s). Most preferably,calcification is cardiovascular calcification.

Cardiovascular calcification as used herein may be understood in thebroadest sense as the formation of one or more plaque(s) localized inone or more blood vessel(s), more preferably at the tunica intima and/orthe tunica media. Plaque formation in a blood vessel may also bedesignated as the formation of intravascular plaque. This intravascularplaque may be one reason for the calcification of blood vessels in thebody of the patient designated as “cardiovascular calcification”.Cardiovascular calcification, thus, the formation of calcified lesionsin the cardiovascular system, may increase the risk of numerous diseasessuch as hypertonia, myocardial infarction and cerebral apoplexy(stroke). Highly pronounced forms of calcification may further lead tothe necessity of amputation of an extremity such as a leg. One severeform of pathological cardiovascular calcification is “Monckeberg'satherosclerosis”, also called “medial calcific sclerosis”. Here, thevessels harden as calcium deposits form in the middle layer of the wallsof medium sized vessels (tunica media). Consequently, the pulse pressureis pathologically increased. Plaque formation may be characterized byfatty streaks. These fatty streaks are typically characterized by anaccumulation of fatty acids, cholesterol and/or one or more othersteroid(s). Often, these plaque formations (plaques) are furthercharacterized by the incorporation of low-density lipoprotein (LDL)and/or white blood cells, especially macrophages that have taken upoxidized low-density lipoprotein (LDL). After these white blood cellshave accumulated large amounts of cytoplasmic membranes they are alsodesignated as foam cells. In a later stage of plaque formation, anextracellular lipid core may be formed. Further, the outer and/or olderportions of the plaque may become more calcific, less metabolicallyactive and more physically stiff over time. The plaque may furthercomprise various amounts of cellular debris, calcium and/or fibrousconnective tissue. Optionally it may form a fibrous cap. Finally, plaquemay potentially lead to platelet clotting, atherosclerosis and/orstenosis.

In the context of the present invention, calcification in vivo may alsobe understood as calcification in the lumen of certain organs such asthe kidney, in particular in the renal pelvis, the ureter, the bladder,the gallbladder and/or the bile duct. As a result, a kidney stone(nephrolith) and a gallstone, respectively, may occur. Kidney stones andgallstones frequently occur throughout the population and may lead tosevere symptoms. Moreover, calcification may occur in soft tissues andmay cause soft tissues to harden. Here; calcification may lead to apathological tissue and potentially to a corresponding pathologicalcondition including potentially severe clinical symptoms. Several typesof tumours such as certain types of brain tumours bear a distinctivelocal calcification (calcium spots), though calcium spots of differentsizes may also occur in the brain without provoking any clinicalsymptoms or indicating any pathological condition.

As used in the context of the present invention, calcification may alsobe calcification associated with any deposit mainly composed of one ormore organic material(s). Exemplarily, this deposit may be, e.g., akidney stone, a gallstone, plaque formation and/or a precursor of onethereof. Preferably, these deposits may be plaque formation.

Alternatively, calcification is calcification in vitro. As used herein,the terms “in vitro” and “ex vivo” may be understood interchangeably.Calcification in vitro may occur in any environment outside the body.Exemplarily, calcification in vitro in the context of the presentinvention may occur in any hollow device getting in contact with afluid, preferably an aqueous fluid. In a medicinal context, said hollowdevice may exemplarily be a needle, an acus, a tube, a syringe, adialysis machine, a dialysis membrane, or a blood or plasmapreservation. Alternatively, said hollow device may also be a tube, apipe (e.g., a water pipe), a washing machine, a dishwasher, a waterboiler, a kettle, a pot a pan, a coffee machine, a tea machine, awashing basin, a bath tub, a toilette, an urinal or any machine or partof a machine getting in contact with a calcifying fluid.

As used in the context of the present invention, the term “fluid” may beunderstood in the broadest sense as any fluid that may lead tocalcification. As used herein, the terms “fluid”, “liquid” and“solution” may be understood interchangeable. Preferably, the fluid isan aqueous fluid, thus, a fluid comprising more than 50% (w/w) water,more than 60% (w/w) water, more than 70% (w/w) water, more than 80%(w/w) water, more than 90% (w/w) water, or more than 95% (w/w) water.The fluid may be a body fluid or may be a technical fluid.

As used in the context of the present invention, the term “propensity ofa fluid for calcification” may be understood in the broadest sense asthe tendency of a fluid to bear calcification. The propensity forcalcification may also be understood in the broadest sense as thecalcification pressure and, therefore, the potency of a fluid to bearcalcification. Therefore, in case the propensity of a fluid forcalcification is high, the fluid will likely show calcification uponincreasing the calcium and/or phosphate concentration, whereas in casethe propensity of a fluid for calcification is low, the fluid willlikely not show calcification or show very little calcification uponincreasing the calcium and/or phosphate concentration. In the context ofthe present invention, the propensity of a fluid for calcification ispreferably the overall propensity of a fluid for calcification, thus,the propensity for calcification mediated by the fluid as a whole in amacroscopic view.

The propensity of a fluid for calcification is the opposite of thepropensity of a fluid to prevent calcification. Consequently, thepresent invention also refers to a method for determining the propensityof a fluid for the prevention of calcification. In this context, theprevention or inhibition of calcification may also be designated as“humoral line of defense”. This humoral line of defense may beoverwhelmed by increased calcium and/or phosphate concentrations.

As used herein the term “determining” may be understood in the broadestsense as the characterization of the state and strength of thepropensity of a fluid for calcification. In this context, the term“determining” may optionally include but may not be limited to any oneof the steps of examining, testing, measuring, investigating, assaying,exploring and assessing the propensity of a fluid for calcification. Inthe context of determining the propensity of a fluid for calcificationin a sample obtained from a patient, said determining may be also beunderstood interchangeably with the term “diagnosing”.

As used in the context of the present invention, the term “sample ofsaid fluid” may be understood in the broadest sense as any compositioncomprising the fluid. In the context of the present invention, the terms“sample of said fluid”, “sample of the fluid” and “sample” may beunderstood interchangeably. Preferably, the sample is a fluid sample. Asused herein, the terms “liquid” and “fluid” may be understoodinterchangeably. Most preferably, the sample is an aqueous sample. Thesample may comprise less than 5% (v/v), more than 5% (v/v), more than10% (v/v), more than 20% (v/v), more than 30% (v/v), more than 40%(v/v), more than 50% (v/v), more than 60% (v/v), more than 70% (v/v),more than 80% (v/v), more than 90% (v/v) of the fluid or may even becomposed of fluid only. Preferably, the sample may comprise between 25%(v/v) and 75% (v/v) fluid, most preferably comprise between 30% (v/v)and 60% (v/v) fluid. However, the person skilled in the art will noticethat the amount of fluid in the sample will depend on the type of fluid,the applied read out method as well as the calcium and phosphateconcentration in the sample.

The term “adding a soluble calcium salt and a soluble phosphate salt”may be understood in the broadest sense as the addition of calcium andphosphate ions to the sample.

As used herein, the term “soluble calcium salt” refers to any saltcomprising calcium cations (Ca²⁺) that is soluble in water.

As throughout the present invention, the term “soluble salt” may beunderstood in the broadest sense as a salt having a solubility of morethan 10 nM, more than 0.1 more than more than 10 more than 0.1 mM, morethan 1 mM or more than 10 mM in water at 20° C.

As used herein, the term “soluble calcium salt” refers to any solublesalt comprising calcium cations (Ca²⁺). Exemplarily, the soluble calciummay be calcium chloride (CaCl₂). The person skilled in the art willunderstand that the applied concentration of calcium cations will dependon the amount of fluid in the sample, the type of the fluid, the appliedread out method as well as the phosphate concentration in the sample.Preferably, calcium ions are added to the sample as an aqueous solutionin water or in an appropriate buffer, wherein soluble calcium salt issoluble, such as, e.g., in Hepes buffer at extensively neutral pH.Alternatively, a calcium salt may also be dissolved directly in thesample of the fluid.

As used herein, the term “soluble phosphate salt” refers to any solublesalt comprising at least one of phosphate (PO₄ ³⁻), hydrogenphosphate(HPO₄ ²⁻) and/or dihydrogen phosphate (H₂PO₄ ⁻) cations. Exemplarily,the soluble phosphate salt may be sodium hydrogen phosphate (Na₂HPO₄)and/or sodium dihydrogen phosphate (NaH₂PO₄). Preferably, the solublephosphate salt is added to the sample as an aqueous solution in water orin an appropriate buffer, wherein also calcium cations are soluble, suchas, e.g., in Hepes buffer at extensively neutral pH. Alternatively, thesoluble phosphate salt(s) may also be dissolved directly in the sampleof the fluid.

In fact, the used concentrations of soluble calcium and phosphate saltsare chosen in a range were differences are well observable betweendifferent samples. Therefore, the overall concentrations of calcium andphosphate, i.e., the concentrations of intrinsic and added calcium andphosphate in sum, may preferably exceed the solubility product ofcalcium phosphate in water or buffer. On the other hand, said overallconcentrations of calcium and phosphate should preferably not exceed themaximal capacity of the precipitation-inhibiting properties of the fluidcontained in the sample by more than five orders of magnitude, more thanfour orders of magnitude, more than three orders of magnitude, more thantwo orders of magnitude or more than one order of magnitude. Preferablycalcium and phosphate are added to the sample independently in order toavoid the formation of calcium phosphate precipitates in a solutionbefore getting in contact with the sample.

Preferably, the concentrations of calcium and phosphate may besupersaturated in the sample. Thus, the product of calcium×phosphate issupersaturated in the sample.

Beside the fluid and the soluble calcium and phosphate salts, the samplemay further comprise any other inorganic and/or organic components whichdo not disturb the method. The sample may further comprise a bufferwhich does not disturb the method such as, e.g., Hepes buffer. The pHmay be adjusted at any pH. Preferably, the pH may be at an extensivelyneutral pH, i.e., between pH 5.5 and pH 8.0, more preferably between pH6.0 and pH 8.0, even more preferably between pH 6.5 and pH 7.8, evenmore preferably between pH ix) 7.2 and pH 7.6, most preferably at aphysiological pH, i.e. at a pH at approximately pH 7.4.

The sample may be admixed manually or automatically. The sample volumemay depend on the applied detection method and will typically varybetween the nanoliter range for small microarray setups up to themilliliter range for macroscopic arrays such as, e.g., centrifugation-or column-based methods.

The samples will typically be used directly obtained from their source(e.g., from a patient or from a technical device) or within few minutesor few days. When the samples are stored for more than few hours, thesamples may preferably be stored at room temperature or in the fridge.Alternatively, the samples may also be stored in a frozen, deep-frozenor freeze dried state and may then be stored at any temperature belowthe freezing point, such as, e.g., at −20°, −80° C. or in liquidnitrogen. A freeze-dried powder may also be stored at ambienttemperature.

As used herein, the term “incubating” may be understood in the broadestsense as subjecting the sample of the fluid to conditions allowing theformation of CPPs.

As used herein, the term “conditions allowing the formation of CPPs”refers to any conditions in which CPPs may be formed. Typically saidCPPs may comprise poorly soluble calcium phosphate.

Preferably, the sample may be incubated at an extensively neutral pH,i.e., at conditions between pH 5.5 and pH 8.0, more preferably betweenpH 6.0 and pH 8.0, even more preferably between pH 6.5 and pH 7.8, evenmore preferably between pH 7.2 and pH 7.6, most preferably at aphysiological pH, i.e. at a pH at approximately pH 7.4.

As a precipitation reaction will typically be temperature-sensitive, forcomparison of different samples with another and/or for comparison witha calibrating curve, the temperature or temperature profile is set to bein a comparable range. Most preferably, the sample is incubated at aconstant temperature and the difference in temperature between samplesto be compared with another should be less than 5° C., less than 4° C.,less than 3° C., less than 2° C., less than 1° C., less than 0.5° C.,less than 0.25° C. or even less than 0.1° C. Preferably, the constanttemperature may be in the range of from 0° C. to 100° C., morepreferably in the range of from 0° C. to 45° C., even more preferably inthe range of from 4° C. to 42° C., even more preferably in the range offrom 20° C. to 40° C., even more preferably in the range of from 36° C.to 38° C., even more preferably in the range of from 36.0° C. to 37.5°C. and most preferably in the range of from 36.5° C. to 37.0° C.

Alternatively, the temperature may be varied during the measurement inthe form of a temperature profile. This temperature profile may also becomparable in all samples to be compared with another.

As use in the context of the present invention, the terms “calciproteinparticle”, “calcium protein particle”, “calcified protein particle” and“CPP” may be understood in the broadest sense as any particle comprisingcalcium cations. The terms “protein”, “polypeptide” and “peptide” may beunderstood interchangeably throughout the invention. In the context ofthe present invention, the terms “particle”, “nanosphere”,“precipitate”, “colloid” and “aggregate” may be understoodinterchangeably. In the context of the formation of CPPs, the particlesare preferably formed by the accumulation of molecules in a samplesolution. Preferably, in the CPPs, the calcium cations form a poorlysoluble salt. Exemplarily said poorly soluble salt may be calciumphosphate and/or one or more complex(es) thereof. Optionally, saidpoorly soluble salt may form crystals, preferably crystals in thenanometer or micrometer range, most preferably in the nanometer range.The regular crystal structure may optionally interrupted by theincorporation of one or more polypeptide(s) or other molecules. However,the CPP may further comprise any other inorganic or organic molecule(s)and/or ion(s) found in the fluid and/or added to the sample such as,e.g., one or more polypeptide(s) that may interact with calcium (e.g., afetuin polypeptide (e.g., fetuin-A), an albumin polypeptide (e.g., serumalbumin), calbindin, S-100 protein(s), osteocalcin, vitamin D dependentcalcium binding protein, lactoferrin, lactoferricin), organic anions(e.g. oxalate), inorganic anions (e.g., carbonate and/or pyrophosphate),organic cations, inorganic cations (e.g., magnesium and/or H⁺ ions),vitamins (e.g., vitamin D), hormones (e.g., steroids, thyroxin). It willbe understood by a person skilled in the art that a CPP may in generalpreferably comprise components interacting with calcium anions and/orcalcium salts and other components interacting with said componentsinteracting with calcium anions and/or calcium salts. However, othermolecules, in particular hydrophobic molecules, may also accumulate in aprecipitate.

In the context of the present invention, a CPP is preferably a solid orsemi-solid particle. Therefore, the formation of CPPs in a fluid sampleleads to a suspension. A CPP may have any size. Preferably, a CPP has asize in the nanometer or micrometer range. As used in this context, theterm “size” means the average diameter of the precipitates.

Optionally, the CPPs may further be fluorescently stained, such as e.g.,by admixing a fluorescent polypeptide (e.g., cyan fluorescent protein(CFP), green fluorescent protein (GFP) or yellow fluorescent protein(YFP), red fluorescent protein (RFP), mCherry, etc.), a small-moleculedye (e.g., a Cy dye (e.g., Cy3, Cy5, Cy5.5, Cy 7), an Alexa dye (e.g.,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 680,Alexa Fluor 750), a Visen dye (e.g. VivoTag680, VivoTag750), an S dye(e.g., S0387), a DyLight fluorophore (e.g., DyLight 750, DyLight 800),an IRDye (e.g., IRDye 680, IRDye 800), a fluorescein dye (e.g.,fluorescein, carboxyfluorescein, fluorescein isothiocyanate (FITC)), arhodamine dye (e.g., rhodamine, tetramethylrhodamine (TAMRA)) or aHOECHST dye), a quantum dot or a combination of two or more thereof.However, preferably, the CPPs are not fluorescently stained.

Depending on its components, a CPP of the present invention may be anextensively spherical particle, a spindle shape particle, a particle ofrandom shape or a crystalline particle. Primary CPPs are extensivelyspherical (cf. FIG. 5A), whereas a secondary CPPs bear a spindle orrandom shape (cf. FIG. 5B). Depending on the used method, the differentshape may or may not have influence on the light scattering propertiesof the particle. Preferably, it has influence on light scattering.

In the context of CPPs, the person skilled in the art will discriminatebetween primary and secondary CPPs (cf. Jahnen-Dechent et al., 2011).The primary CPPs have a smaller diameter than the secondary CPPs.Further, the primary CPPs bear an extensively spherical shape, whereasthe secondary CPPs bear a rather random shape.

In this context, the terms “primary calciprotein particles” and “primaryCPPs” may be understood in the broadest sense as particles that areinitially be formed within a short time of few seconds or few minutesafter adding the soluble calcium salt and the soluble phosphate salt tothe sample of the fluid. The average diameter of the primary CPPs isbelow 100 nm. Preferably, the size of primary CPPs will be in the rangeof between 50 nm and 100 nm, most typically in the range of about 60 nmto 75 nm. However, it will be understood by a person skilled in the artthat the size will also depend on the specific concentrations andconditions used in the method. Typically, as protein components, primaryCPPs mainly comprise fetuin-A and albumin.

In this context, the terms “secondary calciprotein particles” and“secondary CPPs” may be understood in the broadest sense as particlesthat are formed upon incubation by the spontaneous transition of primaryCPPs in a transitional ripening step. This step may also take place in atimed and coordinated manner. Typically, this transition will also beassociated with an increase in particle diameter (Wald et al., 2011).This transition step may require considerably more time than the initialformation of the primary CPPs. The person skilled in the art will noticethat the transition rate depends on the reaction conditions. Typically,the extensive completeness of the transition step may require time inthe range of several minutes, several hours, several days, several weeksor even several months. The extensive completeness of the transitionstep may require more than 1 min, more than 5 min, more than 10 min,more than 30 min, more than 1 h, more than 2 h, more than 6 h, more than12 h, more than one day, more than two days, more than one week, morethan one month, more than six months or even more than one year. Theaverage diameter of the secondary CPPs is larger than 100 nm. Typically,the secondary CPPs may have a size in the range of between 100 nm and500 nm, most typically in the range of about 100 nm to 200 nm. However,it will be understood by a person skilled in the art that the size willalso depend on the specific concentrations and conditions used in themethod. Typically, as protein components, secondary CPPs comprisefetuin-A, albumin and further high and low molecular weight components.Preferably, these further protein components have a molecular mass ofbetween 5 and 150 kDa, more preferably of between 10 and 100 kDa.Further, high-molecular weight complexes may occur having a molecularmass of larger than 150 kDa.

As used in the context of the present invention, the term “rate of theformation of primary CPPs” may be understood in the broadest sense asthe rate of the generation of primary CPPs in a fluid over time. Thismay be exemplarily quantified as the amount of primary CPPs, as thevolume of primary CPPs or as the mass of primary CPPs in a specificfluid or sample volume. Typically, the primary CPPs will be formedinitially in the fluid or sample and may disappear or diminish in amountupon by the transition of primary CPPs into secondary CPPs. Theformation of primary CPPs will typically depend on the calcium, thephosphate, the magnesium and the proton concentration of the fluid aswell as the concentration of calcification inhibitors and promoters.

As used in the context of the present invention, the term “rate of theformation of secondary CPPs” may be understood in the broadest sense asthe rate of the generation of secondary CPPs in a fluid over time. Thismay be exemplarily quantified as the amount of secondary CPPs, as thevolume of secondary CPPs or as the mass of secondary CPPs in a specificfluid or sample volume. Typically, the secondary CPPs will be formed bythe transition of primary CPPs into secondary CPPs. Like the formationof primary CPPs, also the formation of secondary CPPs will typicallydepend on the calcium, the phosphate, the magnesium and the protonconcentration of the fluid as well as the concentration of calcificationinhibitors and promoters.

As used in the context of the present invention, the term “determiningthe rate of the formation” may be understood in the broadest sense asthe observance and quantification of alterations in the generation ofCPPs. As indicated above the formation of CPPs may typically lead to anincreased turbidity of the solution. The absorption of the solution andthe light scattering properties of the solution increase. The formationrate may be determined by any means known in the art. Exemplarily, itmay be determined my means of any method known in the art.

As used in the context of the present invention, the term “amount ofprimary and/or secondary CPPs” refers to the number of primary and/orsecondary CPPs comprised in a specific volume of the fluid or thesample, the mass of the primary and/or secondary CPPs comprised in aspecific volume of the fluid or the sample or the volume of the primaryand/or secondary CPPs comprised in a specific volume of the fluid or thesample. The mass of primary and/or secondary CPPs comprised in aspecific volume may be determined as the mass of the dry particles orthe mass of the particles in solution.

As used in the context of the transition of primary CPPs into secondaryCPPs, the term “transition” may be understood in the broadest sense asthe rearrangement of the calcium-comprising particles, i.e., thecalciprotein particles (CPPs), upon incubation, namely, the conversionof one type of CPPs, i.e., primary CPPs, into another type of CPPs,i.e., secondary CPPs. Herein, the terms “transition”, “conversion” and“rearrangement” may be understood interchangeably. The transition mayalso be understood as a transitional ripening step. This step may takesplace in a timed and coordinated manner. Typically, the transition willbe associated with an increase in particle diameter.

As used in the context of the present invention, the term “transitionrate” may be understood in the broadest sense as the kinetic of thetransition of primary CPPs into CPPs. Typically, the transition rate isquantified as the time point of the half maximal transition time (T₅₀)of the transition of primary CPPs into secondary CPPs. In the context ofthe present invention, it is demonstrated that the transition ratepreferably is delayed in the presence of magnesium (Mg²⁺) andaccelerated in the presence of phosphate (PO₄ ³⁻, HPO₄ ²⁻, H₂PO₄ ⁻) (cf,FIG. 2A).

As used in the context of the present invention, the term “determiningthe transition rate” may be understood in the broadest sense as theobservance and quantification of alterations in the average size ofCPPs. As mentioned above, in the context of the present invention,transition will typically mean an increase in size of the CPPs. Thetransition rate may be determined by any means known in the art.Exemplarily, it may be determined my means of an optical method (e.g.,measuring the absorption, detecting changes in light scattering,detecting changes in laser diffraction and/or correlation spectroscopy).

As used in the context of the present invention, the term “increasedformation rate” may be understood in the broadest sense as theoccurrence of a faster formation of primary and/or secondary CPPs.

As used in the context of the present invention, the term “increasedamount” may be understood in the broadest sense as the occurrence of ahigher number, amount or volume of primary and/or secondary CPPs.

As used in the context of the present invention, the term “increasedtransition rate” may be understood in the broadest sense as theoccurrence of a faster transition of primary CPPs into secondary CPPs.In this context, the transition rate T₅₀ determined for a sample of thefluid may be compared with a one or more control sample(s).

As used herein, the term “increased propensity for calcification” may beunderstood in the broadest sense as the occurrence of a higherpropensity of a fluid for calcification. Therefore, a fluid withincreased propensity for calcification will have a higher tendency toprecipitate insoluble or poorly soluble calcium salt(s). Thus, increasedpropensity for calcification also means that the likelihood of theoccurrence of calcification and/or the intensity of calcification isincreased.

Preferably, step (iii) of the method of the present invention at leastcomprises determining the transition rate of the transition of primaryCPPs into secondary CPPs (step (c)).

In a preferred embodiment, the step of determining one or more of therate of the formation of primary and/or secondary CPPs, the amount ofprimary and/or secondary CPPs and/or the rate of the transition ofprimary CPPs into secondary CPPs (step (iii)) is performed by an opticalmethod, in particular an optical method selected from the groupconsisting of:

absorptiometry,detection of light scattering,correlation spectroscopy,or a combination of two or more thereof.

As used in the context of the present invention, the term “opticalmethod” may be understood in the broadest sense as any method whereinlight is used for the detection, characterization and/or quantificationof primary and secondary CPPs. The optical method may be a visual methodsuch as e.g., a microscopic method, or may be a non-visual method suchas, e.g., the detection of light scattering or laser diffraction orcorrelation spectroscopy.

As used in the context of the present invention, the term“absorptiometry” may be understood in the broadest sense as thedetermination of the absorption or extinction of the fluid of thepresent invention. Typically, the absorption of the solution willincrease upon the formation of primary and secondary CPPs as theturbidity increases. This turbidity may be assessed by the naked eye baycomparison with one or more calibration sample(s) or may be measured bya technical device. Also the transformation of primary CPPs intosecondary CPPs may have an influence on the absorption. Absorptiometrymay be performed by any device known in the art such as, e.g., aphotometer using a cuvette or a plate reader using a microtiter plate(e.g., a 96-well plate or a 386-well plate). Absorptiometry may beperformed using light of any wavelength suitable in the art. Preferably,the wavelength will be in the range of between 100 nm and 2000 nm, morepreferably in the range of between 200 nm and 1500 nm, even morepreferably in the range of between 250 nm and 1000 nm, more preferablyin the range of between 280 nm and 1000 nm, more preferably in the rangeof between 300 nm and 900 nm, more preferably in the range of between400 nm and 800 nm. The person skilled in the art will recognize that thechosen wavelength is preferably in the range where neither the cuvetteor microtiter plate, nor the sample fluid itself shown a considerableabsorption.

As used throughout the invention, the wavelength of the excitation lightmay be chosen by the choice of a particular light source (e.g., a laserbearing specific laser lines), by conduction of the excitation lightthrough one or more filter(s), one or more semipermeable mirror(s)and/or one or more acusto-optical beam splitter(s) and/or by splittingthe light into particular wavelength(s) by means of one or more prism(s)and/or one or more optical grid(s). Likewise, the light passed thesample may be conducted through one or more filter(s), one or moresemipermeable mirror(s) and/or one or more acusto-optical beamsplitter(s) and/or by splitting the light into particular wavelength(s)by means of one or more prism(s) and/or one or more optical grid(s) inorder to enable the detection of one or more specific wavelength(s).

Light may be detected by any device known in the art. Exemplarily, thedetector(s) may be avalanche photo diode(s) (APD(s)), a photomultipliertune(s) (PMT(s)) and/or a diode array. A diode array may comprise morethan four, more than ten, more than 100, more than 1000 or even morethan 10000 diodes.

As used in the context of the present invention, the term “lightscattering” may be understood in the broadest sense as any form ofscattering in which light is the form of propagating energy which isscattered. Light scattering is a phenomenon well-known by those skilledin the art. Therefore, light scattering may be the deflection of a rayfrom a straight path. Exemplarily, it may be provoked by irregularitiesin the propagation medium. In the context of the present invention, saidirregularities in the propagation medium are preferably caused by theCPPs in the sample and the interface between the sample fluid and saidCPPs. The scattering detected in the context of the present invention ismainly a diffuse scattering due to the random and dense distribution ofthe CPPs throughout the sample. The shape of the particles may haveinfluence on the light scattering properties of the particles. The lightscattering may further depend on the wavelength of the light beingscattered, therefore, on the frequency of the excited light. As usedherein, the terms “excited light” and “incident light” may be understoodinterchangeably.

As used in the context of the present invention, the term “detection oflight scattering” may be understood in the broadest sense as any methodknown in the art for the identification, determination andquantification of scattered light. Typically, a light beam of a light ofany light source is excited into the sample and the light scattered inone, two or more angle(s) to the irradiating light beam is detected.

Detection of light scattering may be performed by any method known inthe art. Exemplarily, detection of light scattering may be detection ofRayleigh scattering, Raman scattering, Mie scattering, Tyndallscattering, Brillouin scattering. Preferably, light scattering is mainlyRayleigh scattering.

In the context of the present invention, Rayleigh scattering is theelastic scattering of light by the particles of the CPPs, wherein thewavelength of the scattered light typically is smaller than thewavelength of the excited light. The intensity of Rayleigh scattering isdependent on the size of the particles. Typically, it is extensivelyproportional the sixth power of their diameter and extensively inverselyproportional to the fourth power of the wavelength of the excited light.The wavelength of the used excitation light will typically be at acomparably short wavelength below 400 nm, below 350, below 300 nm, below250 nm, below 200 nm or even below 150 nm. However, a person skilled inthe art will notice that regarding to the experimental setup also otherwavelengths may be applicable.

Raman scattering is well-known by those skilled in the art and bases oninelastic light scattering, wherein the light interacts with opticalphonons, which are predominantly intra-molecular vibrations androtations. Standard spectrometers using scanning monochromators may beused to measure Raman scattering. Mie scattering is a broad class ofscattering of light by spherical particles, in particular sphericalparticles much larger than the wavelength of the excited light. Thescattering intensity is generally not strongly dependent on thewavelength, but is sensitive to the particle size. Mie scatteringintensity for large particles is extensively proportional to the squareof the particle diameter. Tyndall scattering is similar to Miescattering without the restriction to spherical geometry of theparticles. It is particularly applicable to the colloidal suspensions ofthe present invention. Brillouin scattering occurs from the interactionof photons with acoustic phonons in solids, which are vibrational quantaof lattice vibrations, or with elastic waves in liquids.

More preferably, the sample is excited by a laser beam and detection ofthe scattered light is measured in one, two or more angle(s). Mostpreferably, light scattering is detected by nephelometry.

The forward scattered light (measured at an angle of between 160° and180°) and/or the sideward scattered light (measured at an angle of<160°) may be detected. Herein, forward scattered light may be lightdetected at an angle of approximately 1800° referred to the excitedlaser beam, at an angle of approximately 170° referred to the excitedlaser beam or at an angle of between approximately 160° referred to theexcited laser beam. The sideward scattered light may be light detectedat any other angle referred to the excited laser beam. Exemplarily, thesideward scattered light may detected at an angle of approximately 30°,40°, 45°, 70°, 75° and/or 90°. However, the person skilled in the artwill notice that the detection at any other angle may also be used inthe context of the present invention. The detection may also beperformed at varying angles, i.e., at angles varying over time duringthe method. Alternatively, the detection may also be performed at two ormore different angles at the same time. When light scattering indifferent angles is measured, this may be designated as dynamic lightscattering. The excitation may be continuous excitation or may be pulsedexcitation, wherein the excitation time is in the range or nanoseconds,microseconds or seconds. Pulsed excitation may be repeated any number oftimes. The measurement may be performed once or more than once.Exemplarily, the measurement may be repeated two times, three times,four times, five times, ten times, 20 times, 30 times, 40 times, 50times, 100 times, 150 times, 200 times, 250 times or more often.Exemplarily, one measurement cycle may last less than 1 μs,approximately 10 μs, 0.1 s, 0.5 s, 1.0 s, 1.5 s, 2 s, 5 s, 10 s orlonger.

The wavelength and laser intensity may be adjusted in accordance withthe sample. As used herein, detection of laser diffraction may beperformed by any method known in the art suitable for the detection oflight diffraction. In general, light diffraction bases on quantumtheory. The wavelength associated with a particle is the de Brogliewavelength:

momentum of the particle(p)=[Planck's constant(h)]/[wavelength(λ)]

Herein, for slow-moving particles, the momentum of the particlecorresponds to its mass multiplied by its velocity.

As used in the context of the present invention, sedimentationtechniques may be understood in the broadest sense as any methods basedon the sedimentation of the CPPs of the present invention. Asedimentation technique may be based on natural gravity only or may beexpedited by centrifugal force. Preferably, a sedimentation technique inthe context of the present invention may be expedited by centrifugalforce. Optionally, the volume and/or the weight of the precipitate maybe determined, in particular the volume ratio and/or weight ratio of theprecipitate and the supernatant may be determined. Alternatively and/oradditionally, the sedimentation constant of the precipitates may bedetermined. Alternatively and/or additionally, the weight density of theprecipitates may be determined, such as, e.g., by sucrose gradientcentrifugation.

It will be understood that two or more of the methods described abovemay be combined with another. Further, it will also be understood thattwo or more of the methods described above may be combined with one ormore optical methods.

The excitation light may be any light source known in the art.Preferably, the excitation light is a laser beam or an Hg-lamp.

In a preferred embodiment, the excitation light used in the opticalmethod is a laser beam.

As used in the context of the present invention, a laser may be anylaser known in the art. A laser typically shows a comparably high degreeof spatial and temporal coherence, unattainable using othertechnologies. Preferably, the laser light is further monochromaticand/or one laser line is selected for emission. Exemplarily, the lasermay be a HeNe or an argon ion laser. A HeNe laser typically emits lightof a wavelength of approximately 633 nm, an argon ion laser typicallyemits light of a wavelength of approximately 488 nm. Laser intensity maybe adjusted to the sample. For nephelometry, laser intensity mayexemplarily be in the range of from 0.1 to 1000 mW, preferably in therange of from 1 to 100 mW, more preferably in the range of from 10 to 50mW.

Further, the light emitted by the laser and/or the scattered light maybe conducted through one or more filter(s), one or more semipermeablemirror(s) and/or one or more acusto-optical beam splitter(s) and/or maybe splitted into particular wavelength(s) by means of one or moreprism(s) and/or one or more optical grid(s). Light may be detected byany device known in the art. Exemplarily, the detector(s) may beavalanche photo diode(s) (APD(s)), a photomultiplier tune(s) (PMT(s))and/or a diode array. A diode array may comprise more than four, morethan ten, more than 100, more than 1000 or even more than 10000 diodes.

In a more preferred embodiment, the optical method is performed bydetecting light scattering, preferably by dynamic light scattering, morepreferably by cross-correlation dynamic light scattering, even morepreferably by three-dimensional cross-correlation dynamic lightscattering, in particular by nephelometry.

As used herein, detecting light scattering may be detecting static lightscattering or detecting dynamic light scattering. Preferably, detectinglight scattering is detecting dynamic light scattering.

As used in the context of the present invention, the term “dynamic lightscattering” may be understood interchangeably with the terms “photoncorrelation spectroscopy” and “quasi-elastic light scattering” astechnique in physics, which can be used to determine the sizedistribution profile of small particles in suspension in solution bymeans of the observance of a time-dependent fluctuation in thescattering intensity. Typically, these fluctuations will occur due tothe fact that the small molecules in solutions are undergoing Brownianmotion and so the distance between the scatterers in the solution isconstantly changing with time resulting in alternating constructive anddestructive interference by the surrounding particles.

Within this intensity fluctuation, information on the time scale ofmovement of the scatterers is obtainable.

The dynamic light scattering may be based on any type of lightscattering. Typically, this light scattering comprises Rayleighscattering. Preferably, the emission light is laser. Exemplarily, thedynamic light scattering may be quasi-elastic laser light scattering.Then, the dynamic information of the particles may be derived from anautocorrelation of the intensity trace recorded during the experiment.

In the context of the present invention, the term “cross correlation”may be understood in the broadest sense as a measure of similarity oftwo waveforms in signal processing. It is commonly used for searching along-duration signal for a shorter feature.

As used herein, the terms “three-dimensional cross-correlation dynamiclight scattering”, “3D cross-correlation dynamic light scattering”, and“3D dynamic light scattering” may be understood interchangeably.

As used in the context of the present invention, the term “nephelometry”may be understood in the broadest sense as a method performed bymeasuring the turbidity in an extensively aqueous sample by measuringthe light intensity of light passing through the sample at an anglebased on the principle that a dilute suspension of small particles willscatter light passed through it rather than simply absorbing it.Nephelometry may be measured manually or in a semi-automated or anautomated manner.

In this context, the scattered light may be measured at any angle suchas, e.g., at approximately 30°, 40°, 45°, 70°, 75° and/or 90°. Mostpreferably, the light is detected in a 90° geometry. Typically, thelight source is a laser. The results obtained for the sample mayoptionally be compared to one or more value(s) obtained for calibrationsamples(s) or may be compared with one or more standard curve(s).

Nephelometry may be end point nephelometry or may be measured over timeas kinetic nephelometry. As used herein, end point nephelometry meansthat the formation or rearrangement is size of the CPPs may beextensively completed and does not or nearly not change any more. Inkinetic nephelometry, light scattering is measured over time. Typically,the measurement of light scattering is started right after the calciumand/or phosphate is added and then continued for a certain time. Kineticnephelometry allows observing changes in the particle size distributionand the average particle size over time. Preferably, nephelometry, asused in the context of the present invention, is kinetic nephelometry.In order to enable the detection of large particles, which may havesettled to the bottom of the sample the sample may optionally be shakenor mixed prior to each measurement. As long as the reagent is constantthe rate of change can be seen as directly related to the amount of thepropensity of the body fluid for calcification.

Alternatively to performing step of determining the transition rate ofthe transition of primary CPPs into secondary CPPs (step (iii)) by anoptical method, it may also be preformed by any other suitable methodknown in the art. Preferably such method is based on detecting theaverage size or the average mass of the particles present in the sample.

In the context of the present invention, the step of determining one ormore of the rate of the formation of primary and/or secondary CPPs, theamount of primary and/or secondary CPPs and/or the rate of thetransition of primary CPPs into secondary CPPs (step (iii)) may also beany other method known in the art, thus, also a non-optical method or acombination of two or more non-optical methods or a combination of oneor more optical method(s) and one or more non-optical method(s).

In an preferred embodiment of the present invention the step ofdetermining one or more of the rate of the formation of primary and/orsecondary CPPs, the amount of primary and/or secondary CPPs and/or therate of the transition of primary CPPs into secondary CPPs (step (iii))is performed by any method selected from the group consisting of:

-   sedimentation techniques,-   filtration analysis,-   size exclusion chromatography,-   granulometry,-   acoustic spectroscopy,-   or a combination of two or more thereof.

As used in the context of the present invention, the term “sedimentationtechnique” may be understood in the broadest sense as any method basedon the deposition of the primary and/or secondary CPPs. Preferably, theprimary CPPs and the secondary CPPs may bear different sedimentationconstants and may, therefore, sediment at different time points.Typically, the secondary CPPs may sediment first. A sedimentationtechnique of the present invention may be based on gravitational forceonly or may also include centrifugal force. Preferably, thesedimentation technique of the present invention may also includecentrifugal force. A pallet comprising the secondary CPPs and/or theprimary CPPs may be formed. Alternatively or additionally, equilibriumcentrifugation (e.g., sucrose gradient centrifugation) may be used.Herein, the primary CPPs and the secondary CPPs may be separated fromanother due to their different mass density. In general, the separatedprimary CPPs and the secondary CPPs may be investigated further bydetermining the volume or mass thereof or by observing these via opticalmethods such as, e.g., microscopy. Optionally, the volume and/or theweight of the precipitate may be determined. In order to observe akinetic profile of the transition of primary CPPs into secondary CPPs, anumber of samples may be investigated.

As used herein, a microscopic method may exemplarily be light microscopy(e.g., brightfield microscopy), fluorescence microscopy (e.g.,(confocal) laser scanning microscopy (LSM), stimulated emissiondepletion microscopy (STED microscopy)), electron microscopy (e.g.,scanning electron microscopy (SEM), transition electronic microscopy(TEM), scanning transmission electron microscopy (STEM), scanningtunneling microscopy (STM)), scanning helium ion microscopy (SHIM),scanning probe microscopy (SPM) and/or atomic force microscopy (AFM).

As used in the context of the present invention, the term “filtrationanalysis” may be understood in the broadest sense as any method based onmolecular and/or particulate sieving of the sample. As used herein, theterms “sieving” and “filtrating” may be understood interchangeably.Filtrating may be dead end or continuous filtration. Optionally,filtration may also be cross-filtration. The particles may be separatedand/or isolated due to their size. Preferably, the filter has a sizeallowing the primary CPPs to pass through, whereas the secondary CPPsare retarded. Optionally, the volume and/or the weight of theprecipitate may be determined, in particular the volume ratio and/orweight ratio of the precipitate and the supernatant may be determined.

As used in the context of the present invention, size exclusionchromatography may be understood in the broadest sense as anychromatographic method suitable for separating the CPPs of the presentinvention from the sample of the body fluid. Preferably, the primaryCPPs and the secondary CPPs show different elution profiles. Sizeexclusion chromatography may also be designated as molecular weightchromatography. Optionally, the volume and/or the weight of theprecipitate may be determined, in particular the volume ratio and/orweight ratio of the particular fractions may be determined.

As used in the context of the present invention, optical granulometrymay be understood in the broadest sense as any method for the opticaldetermination of the size of the particles. In the context of thepresent invention, optical granulometry will typically be a microscopebased method. Optionally, a computer assisted method, preferably basedon a binary mask, may be used in order to identify particular structuredand characterize and quantify these in size and number. The personskilled in the art will know how to apply a corresponding macro of thepresent invention. This macro may be run on any suitable computerprogram such a, e.g., by the open source program ImageJ based on Java.

As used in the context of the present invention, the term “acousticspectroscopy” may be understood in the broadest sense as any methodemploying sound for collecting information on the particles that aredispersed in fluid. In the context of the present invention, this soundtypically will be ultrasound. Dispersed particles will absorb andscatter ultrasound similarly to light. Acoustic spectroscopy may bebased on measuring scattered energy versus angle or measuring thetransmitted energy versus frequency. The resulting ultrasoundattenuation frequency spectra are the raw data for calculating particlesize distribution. One example for acoustic spectroscopy is ultrasoundattenuation spectroscopy. The average size of the particles may bedetermined.

Further, step (iii) of the present invention may also be performed byelectroresistance counting. As used in the context of the presentinvention, electroresistance counting may be understood in the broadestsense as any method based on detecting changes in the electricresistance of the sample. Preferably, the sample is conducted through athin needle of a size allowing only a single particle or few particlespassing through at once. One example of electroresistance counting isthe Coulter Counter, which measures the momentary changes in theconductivity of a liquid passing through an orifice that take place whenindividual non-conducting particles pass through. The particle count isobtained by counting pulses, and the size is dependent on the size ofeach pulse. Preferably, the passage of one of the larger secondary CPPswill show a stronger signal than the passage of one of the smallerprimary CPPs. By setting a certain threshold in size, the number ofprimary and secondary CPPs may be determined.

Preferably, electroresistance counting is Electrochemical ImpedanceSpectroscopy (EIS). As used herein, the terms “Electrochemical ImpedanceSpectroscopy”, “EIS”, “Impedance Spectroscopy” and “dielectricspectroscopy” may be understood in the broadest sense as a method ofmeasuring the dielectric properties of a medium as a function offrequency based on the interaction of an external field with theelectric dipole moment of the sample, often expressed by permittivity.Optionally, the obtained data obtained by EIS may be expressedgraphically in a Bode plot or a Nyquist plot.

It will be understood that two or more of the methods described abovemay be combined with another. Further, it will also be understood thattwo or more of the non-optical method(s) described above may be combinedwith one or more optical method(s).

In a preferred embodiment, at least one optical method is combined withelectroresistance counting. In a more preferred embodiment, a methodbased on light scattering is combined with electroresistance counting.In an even more preferred embodiment, a method based on dynamic lightscattering is combined with electroresistance counting. In a mostpreferred embodiment, nephelometry is combined with ElectrochemicalImpedance Spectroscopy (EIS). This may be performed by any device.Particularly preferably, this is performed by using one or moremicrofluidic device(s).

Preferably, the employed method(s) may include at least one opticalmethod and/or one acoustical method. More preferably, the method of thepresent invention includes at least one optical method.

In the context of the present invention, the fluid may be any fluid thatmay lead to calcification. The fluid may be a body fluid or may betechnical fluid.

In a preferred embodiment, the fluid is a body fluid, in particularwherein the fluid is blood, blood plasma, blood serum, lymph and/orurine.

As used herein, the term “body fluid” may be understood in the broadestsense as any fluid obtainable from a body such as, e.g., blood, urine,cerebrospinal fluid, lymph, saliva and/or one or more secret(s) from anygland(s). The term “body fluid” may also include any fluid obtainablefrom a body after one or more processing step(s) (e.g., centrifugation,filtration, cross filtration, precipitation and/or any other knownmethod for fractioning biological fluids) such as, e.g., blood serum orblood plasma. As used herein, the terms “blood serum” and “serum” may beunderstood interchangeably. Likewise, the terms “blood plasma” and“plasma” may be understood interchangeably. Preferably, the body fluidis blood, blood plasma, blood serum or lymph.

The body fluid will typically be used directly obtained from the patientor within few minutes or few days. When the samples are stored for morethan few hours, the samples may preferably be stored at room temperatureor in the fridge. As used in the context of the present invention, theterms “room temperature” and “ambient temperature” may be understoodinterchangeably. Alternatively, the samples may also be stored in afrozen, deep-frozen or freeze dried state and may then be stored at anytemperature below the freezing point, such as, e.g., at −20°, −80° C. orin liquid nitrogen. A freeze-dried powder may also be stored at ambienttemperature. Obtaining the body fluid from a patient is typically of nohealth risk for the patient and can be also conducted by persons who areno medical experts such as, e.g., even the patient at home.

In a more preferred embodiment, the fluid is a body fluid obtained froma patient, preferably wherein said patient has developed calcificationand/or is at risk of developing calcification, in particular whereinsaid patient is a dialysis patient

Preferably, the patient who has developed calcification and/or is atrisk of developing calcification, has developed calcified plaques and/oris at risk of developing calcified plaques. However, calcification mayalso be microcalcification or may be calcium precipitation in solution,thus, the formation of any calcium precipitates, optionally includingprimary and/or secondary CPPs.

The body fluid may be obtained from a patient or may be obtained from apreserved sample or may be artificial body fluid.

As used herein, the term “obtained from a patient” may be understood inthe broadest sense as receiving a body fluid from the body of a patientby any means. Exemplarily, blood may be collected from a blood vessel,lymph may be corrected from a lymph vessel, cerebrospinal liquid may beobtained from the cerebrospinal lumen and/or urine and/or sweat may becollected. It will be understood that the body fluid may also beprocessed further. Exemplarily, blood plasma or blood serum blood may beextracted from the blood by any means known in the art. The body fluidmay be used in a method of the present invention or may be stored atappropriate conditions for up to one hour, up to two hours, up to sixhours, up to twelve hours, up to a day or even longer. A sample of bodyfluid stored for more than one day may be designated as a preservedsample.

As used herein, the term “preserved sample” may be understood in thebroadest sense as any sample that has been stored for more than morethan one day, more than two days, more than a week, more than two weeks,more than a month, more than two months, more than six months or morethan a year.

The body fluid sample may be stored as the pure body fluid or may bestored as a sample further comprising other components of the sample assummarized in detail above. In general, all storage conditions suitablefor the body fluid may be used. Exemplarily, the body fluid sample maybe stored at room temperature, at 4° C., at −20° C., at −80° C. or inliquid nitrogen. Therefore, the body fluid sample may be stored in aliquid or frozen state.

Alternatively, the body fluid sample may be stored in a freeze-dried ordried state. The preserved sample may exemplarily be a bloodpreservation, a serum or plasma preservation, or a lymph preservation.

The patient may be a healthy patient or a patient who is at particularrisk of calcification. Preferably, the patient bears an increasedpropensity for developing calcified plaques. This increased propensitymay have congenital and/or acquired reason(s).

As used in the context of the present invention, the term “patient” maybe understood in the broadest sense as any subject or individual a bodyfluid is obtainable from, irrespective, clinical symptoms occur or donot occur. The patient may be any animal, including humans. Preferably,the patient is a mammal, most preferably a human.

The patient who has developed calcified plaques and/or who is at risk ofdeveloping calcified plaques may be healthy or may bear a particularrisk of calcification. The patient may or may not suffer from anyclinical symptoms such as, e.g., hypertonia, diabetes, kidneydysfunction or a rheumatoid disease. Exemplarily, kidney dysfunction maybe chronic kidney disease (CKD).

As used herein, the term “body” may be understood in the broadest senseas any living subject or subject that had lived less than ten years ago,less than five years ago, less than four years ago, less than threeyears ago, less than two years ago, less than one year ago, less thansix months ago, less than five months ago, less than four months ago,less than three months ago, less than two months ago, less than onemonth ago, less than two weeks ago, less than one week ago, less thantwo days ago, less than one day ago, less than twelve hours ago, lessthan six hours ago or less than one hour ago. Preferably, the body isthe body of any animal including human. An animal is preferably avertebrate, more preferably an endothermal animal, even more preferablya mammal, most preferably a human.

Optionally, a compound which is a potential or known inhibitor orpromoter of calcification may be added to the body fluid. Then, thepotency of said compound for inhibiting or promoting calcification maybe tested, in particular when a sample comprising said compound iscompared with a corresponding sample without the inhibitor.Advantageously, the method of the present invention provides thepossibility to characterize the action of the compound in its naturalcontext, i.e., in the body fluid. Inhibitors of calcification areexemplarily Fetuin (e.g., Fetuin-A) and albumin (e.g., serum albumin).In vitro, also chelating agents such as, e.g.,ethylenediaminetetraacetic acid (EDTA) or immobilized cation exchangermay be used to inhibit calcification.

Further, in addition or alternatively, phosphate binders may also beused to inhibit calcification. Exemplarily, common phosphate binderscomprise aluminium hydroxide (e.g., Alucaps), calcium carbonate (e.g.,Calcichew, Titralac), calcium acetate (e.g., Phosex, PhosLo), lanthanumcarbonate (Fosrenol), sevelamer (e.g., Renagel, Renvela) and calciumacetate/magnesium carbonate (e.g., Renepho, OsvaRen).

The body fluid may also be an artificial body fluid obtained by admixingand dissolving one or more component(s) is an aqueous solution.

As used herein, the term “dialysis patient” may be understood in thebroadest sense as any patient who needs artificial replacement orsupport for lost or limited kidney function. It will be understood thata dialysis patient is typically a patient of particular risk ofdeveloping calcified plaques.

In another more preferred embodiment, the patient suffers from vascular,valvular and/or soft tissue calcification, preferably wherein saidpatient further suffers from a rheumatoid disease, a malignant diseaseand/or an infectious disease, in particular wherein the patient shows atleast one of the syndromes selected from the group consisting of:

-   renal dysfunction,-   hypertension,-   diabetes mellitus,-   dyslipidemia,-   a lack of adequate mineralization, in particular osteoporosis and/or    osteomalacia, and-   atherosclerosis.

As used in the context of the present invention, the term “rheumatoiddisease” may be understood in the broadest sense as any medical problemaffecting the joints and connective tissue, typically mainly caused byarthritis. Herein, the terms “rheumatoid disease”, “rheumatism”,“rheumatic disorder” and “rheumatic disease” may be understoodinterchangeably. Rheumatoid disease may include but may not be limitedto sclerodermia, fibromyalgia syndrome, ankylosing spondylitis,bursitis, tendinitis, wrist, capsulitis, osteoarthritis, psoriaticarthritis, rheumatic fever, rheumatic heart disease, rheumatoidarthritis, systemic lupus erythematosus, temporal arteritis, polymyalgiarheumatic, tenosynovitis, palindromic rheumatism and myositis. However,rheumatoid disease may also include non-articular rheumatism (e.g.,regional pain syndrome, soft tissue rheumatism).

As used in the context of the present invention, the term “malignantdisease” may be understood in the broadest sense as any malignantdisorder known in the. A malignant disease may be a disorder leading toneoplasia, in particular one or more tumour(s). A malignant disease maytherefore bean oncolytic disorder including cancer. The term “cancer”may be understood in the broadest sense. It may include the occurrenceprimary and secondary tumors, metastases and other kinds of pathologicalneoplasms. However, a malignant disease may also be a non-oncologicdisorder such as, e.g., malignant hypertension, malignant hyperthermia,malignant otitis externa, malignant tertian malaria (typically caused byPlasmodium falciparum) or neuroleptic malignant syndrome.

An infectious disease may be any infections disease known in the art.

As used herein, the term “syndrome” may be understood in the broadestsense as any association of one or more several clinically recognizablefeature(s), sign(s), symptom(s), phenomenum/phenomena and/orcharacteristic(s) that often occur in the context of a particulardisease or disorder, so that the presence of one or more feature(s) mayindicate the presence of the disease or disorder. A syndrome may beobserved by a medical expert, i.e., by a physician or by a nurse and/ormay be observed and potentially reported by the patient.

As used herein, the term “renal dysfunction” may be understood in thebroadest sense as any malfunction of the renal system, in particular thekidney(s). Exemplarily, renal dysfunction may be chronic kidney disease(CKD). Optionally, the renal dysfunction may also be nephropathy. Theterm “nephropathy” as used herein refers to a dysfunction or anon-function of one or both kidneys.

As used herein, the terms “hypertension” and “hypertonia” may beunderstood interchangeably in the broadest sense as a condition of highblood pressure and is well-known in the art.

As used herein, the terms “diabetes mellitus” and “diabetes” may beunderstood in the broadest sense as a group of disorders characterizedby a failure in regulation of the blood sugar level. It comprises agroup of metabolic diseases in which a person has high blood sugar.Typically, diabetes mellitus occurs when the patient's body does notproduce enough insulin and/or when cells in the patient's body do notsuitable respond to the insulin that is produced. Diabetes mellitus mayoptionally lead to numerous clinical and non-clinical symptoms such as,e.g., polyuria (frequent urination), polydipsia (increased thirst)and/or polyphagia (increased hunger).

As used in the context of the present invention, the terms“dyslipidemia” and “dyslipidaemia” may be understood interchangeably inthe broadest sense as any disorder characterized by an abnormal amountof lipids (e.g. cholesterol and/or triglyceride(s)) in the blood.

As used in the context of the present invention, the term “osteoporosis”may be understood in the broadest sense as a disorder characterized by areduction of bone mineral density (BMD), deteriorating of bonemicroarchitecture and/or alteration of the amount and variety ofproteins in bone.

As used in the context of the present invention, the term “osteomalacia”may be understood in the broadest sense as a disorder characterized bythe softening of the bones. It may be caused by defective bonemineralization secondary to inadequate amounts of available phosphorusand calcium and/or may be caused by an overactive resorption of calciumfrom the bone as a result of hyperparathyroidism.

As used herein, the terms “atherosclerosis”, “arteriosclerosis”,“cardiovascular atherosclerosis”, “cardiovascular arteriosclerosis” and“cardiovascular plaque” may be understood interchangeably in thebroadest sense as the deposition of plaque in the blood vessels. Theplaque may be deposited in the lumen of the blood vessels or in the inthe middle layer of the walls of medium sized vessels (tunica media).The plaque may comprise calcium salts in combination with otherminerals, proteins, fatty acids, triglycerides, cholesterols, lowdensity lipoprotein (LDL), high density lipoprotein (HDL) and/or sugars.Furthermore, cells or cellular fragments such as, e.g., macrophages, redblood cells (RBCs) and platelets, may be part of the plaque.Pathological forms of atherosclerosis may be also designated asarteriosclerotic vascular disease or (ASVD). Cardiovascular plaque mayincrease the risk of numerous diseases such as hypertonia, myocardialinfarction and cerebral apoplexy (stroke). Highly pronounced forms ofcalcification may further lead to the necessity of amputation of anextremity such as a leg. One of the most pronounced from ofatherosclerosis is Monckeberg's atherosclerosis. The terms “Monckeberg'satherosclerosis”, “Monckeberg's atherosclerosis” and “medial calcificsclerosis” may be used interchangeably. Monckeberg's atherosclerosis isone of the most severe forms of atherosclerosis. Here, the vesselsharden as calcium deposits form in the middle layer of the walls ofmedium sized vessels (tunica media). As used herein, atherosclerosis mayalso include calciphylaxis, a severe syndrome of vascular calcification,thrombosis and skin necrosis.

In a preferred embodiment, the fluid is an artificial body fluid and/oran infusion solution.

As used herein, the term “artificial body fluid” may be understood inthe broadest sense as any supplement for any body fluid known in theart. Exemplarily, it may be a blood substitute, a plasma substitute or aserum blood substitute. Exemplarily, a blood substitute may comprise butmay not be limited to a perfluorocarbon-based blood substitute (e.g.,Oxygent (Alliance Pharmaceuticals), Oxycyte (Oxygen Biotherapeutics),PHER-O2 (Sanguine Corp), Perftoran), a hemoglobin-based blood substitute(e.g., Hemopure (Biopure Corp), Oxyglobin (Biopure Corp), PolyHeme(Northfield Laboratories), Hemospan (Sangart), Dextran-Haemoglobin(Dextro-Sang Corp), Hemotech (HemoBiotech)), Fluorasol-DA, HemAssist(Baxter International) or Hemolink (Hemosol, Inc.). Further, a bloodsubstitute may be obtained from stem cells, wherein the stem stems arepreferably not human embryonic stem cells. Moreover, dendrimers,biodegradable micelles placental umbilical cord blood or hemerythrin maybe used to obtain a blood substitute.

As used herein, the term “infusion solution” may be understood in thebroadest sense as any solution suitable for infusion known in the art.Exemplarily, an infusion solution may be an isotonic electrolytesolution, isotonic saline, isotonic full electrolyte solution, glucosesolution, Ringer′ solution or a colloidal solution. It will beunderstood that an infusion solution may further bear one or morepharmaceutically active agent(s), one or more pharmaceuticallyacceptable carrier(s) or excipient(s). In general, the infusion solutionmay comprise any pharmaceutically acceptable component, may it becharged or uncharged, polar or nonpolar, a high-molecular weight or asmall molecule.

It will be understood that an artificial body fluid or an infusionsolution may or may not comprise any inhibitor(s) or promoter(s) forcalcification known in the art. It may comprise said inhibitor(s) orpromoter(s) for calcification naturally or said inhibitor(s) orpromoter(s) of calcification may be added to such artificial body fluidor infusion solution. Typically, the technical fluid may comprise anyinhibitor(s) for calcification known in the art. Exemplarily, suchcalcification inhibitor may be a fetuin polypeptide (e.g., fetuin-A) andalbumin (e.g., serum albumin), a chelating agent such as, e.g.,ethylenediaminetetraacetic acid (EDTA), an immobilized cation exchangeror a phosphate binder (e.g., aluminium hydroxide (e.g., Alucaps),calcium carbonate (e.g., Calcichew, Titralac), calcium acetate (e.g.,Phosex, PhosLo), lanthanum carbonate (Fosrenol), sevelamer (e.g.,Renagel, Renvela), calcium acetate/magnesium carbonate (e.g., Renepho,OsvaRen)).

In another preferred embodiment, the fluid is a technical fluid,preferably wherein said fluid is an aqueous fluid, in particular whereinsaid fluid is an industrial process fluid, a fluid comprising a laundryagent or a dishwashing agent, a soap sud, a shower bath, a bathadditive, a cooling fluid, a comestible good or is intended to be usedfor the production of a comestible good.

As used herein, the term “technical fluid” may be understood in thebroadest sense as any fluid that is usable in the context of industrialfacilities, comestible good production and/or domestic purposes. A usein an industrial facility may, e.g., be the use as industrial processfluid, as a general purpose cleaner, as a water additive (e.g., as awater softener) or as cooling agent. A use for comestible goodproduction may comprise cooking, baking, roasting, frying, condensation,thickening, conservation, drying, freeze-drying, infusing, distilling. Acomestible good may comprise, but may not be limited to a beverage(e.g., water, juice, lemonade, an infused drink (e.g., coffee, tea), aninstant beverage (e.g., instant coffee, instant tea), milk, bear, vine,liquor, hard liquor), foodstuff (e.g., bakery goods (e.g., bread, cake,biscuits), sweets, fruit an instant food (e.g., pocket soup). A fluidfor domestic purposes may exemplarily be a laundry agent, a dishwashingagent, a soap sud, a shower bath, a toilet cleaning agent, a softener, arinsing agent, an impregnating agent, a bathroom cleaner, a generalpurpose cleaner, a water additive (e.g., a water softener) or a bathadditive.

The technical fluid may or may not comprise detergents and may or maynot be used to clean laundry, dishes or other domestic goods. Further,the fluid may also be a domestic cleaning agent, a WC cleaning agent, anaquarium cleaning agent or any water additive known in the art toprevent or promote calcification. Alternatively, the technical fluid maybe used in the context of an industrial facility. Typically, thetechnical fluid is intended to avoid calcification in the form ofresiduals comprising calcium salt either in liquid or in dried form.This may be of particular importance as thin tubes or pipes are used.Further, this may be of particular importance as water bearing a highhardness degree is used.

The method of the present invention may be performed at a varyingtemperature, following a particular temperature profile or at a constanttemperature.

As used herein, the term “industrial process fluid” may be understood inthe broadest sense as any fluid used in any industrial process that maybear a propensity for calcification. Preferably, the industrial processfluid may be industrial process water. As used herein, the terms“industrial process water”, “industrial water”, “process water” “servicewater” and “processing water” may be understood interchangeably.Exemplarily, an industrial process fluid may be used as a cooling fluid,as a cleaning fluid, for desalting or as fluid used in a turbine (e.g.,a steam turbine).

It will be understood that a technical fluid may or may not comprise anyinhibitor(s) or promoter(s) for calcification known in the art. It maycomprise said inhibitor(s) or promoter(s) for calcification naturally orsaid inhibitor(s) or promoter(s) of calcification may be added to suchtechnical fluid. Typically, the technical fluid may comprise anyinhibitor(s) for calcification known in the art. Exemplarily, suchcalcification inhibitor may be a fetuin polypeptide (e.g., fetuin-A) andalbumin (e.g., serum albumin), a chelating agent such as, e.g.,ethylenediaminetetraacetic acid (EDTA), an immobilized cation exchangeror a phosphate binder (e.g., aluminium hydroxide (e.g., Alucaps),calcium carbonate (e.g., Calcichew, Titralac), calcium acetate (e.g.,Phosex, PhosLo), lanthanum carbonate (Fosrenol), sevelamer (e.g.,Renagel, Renvela), calcium acetate/magnesium carbonate (e.g., Renepho,OsvaRen)).

In a preferred embodiment, said method is performed at a constanttemperature and/or at a constant pH.

As used herein, the term “constant” means that the temperature remainsextensively the same during step (iii) of the method of the presentinvention. Most preferably, the difference in temperature betweensamples to be compared with another should be less than 5° C., less than4° C., less than 3° C., less than 2° C., less than 1° C., less than 0.5°C., less than 0.25° C. or even less than 0.1° C. Preferably, theconstant temperature may be in the range of from 0° C. to 100° C., morepreferably in the range of from 0° C. to 45° C., even more preferably inthe range of from 4° C. to 42° C., even more preferably in the range offrom 20° C. to 40° C., even more preferably in the range of from 36° C.to 38° C., even more preferably in the range of from 36.0° C. to 37.5°C. and most preferably in the range of from 36.5° C. to 37.0° C.Optionally, the used device may comprise a temperature sensor.

As used herein, the pH (potentia hydrogenii) may be understood ascommonly understood in the art, i.e., as the negative decimal logarithmof the hydrogen ion activity in a fluid.

As used herein, the term “constant” means that the pH remainsextensively the same during step (iii) of the method of the presentinvention. Most preferably, the difference in pH between samples to becompared with another should be less than 3 pH units, less than 2 pHunits, less than 1 pH unit, less than 0.8 pH units, less than 0.6 pHunits, less than 0.4 pH units, less than 0.2 pH units, less than 0.1 pHunits or less than less than 0.05 pH units. The pH may be adjusted atany pH. Preferably, the pH may be at an extensively neutral pH, i.e.,between pH 5.5 and pH 8.0, more preferably between pH 6.0 and pH 8.0,even more preferably between pH 6.5 and pH 7.8, even more preferablybetween pH 7.2 and pH 7.6, most preferably at a physiological pH, i.e.at a pH at approximately pH 7.4.

As mentioned above, the sample is preferably a fluid sample. The methodmay be performed in any device suitable to perform the method of thepresent invention. It will be understood that the choice of the carrierwill depend on the specific method. Exemplarily, the method may beperformed using one or more cuvette(s), using one or more microscopicslide(s), using a multiwell format, using a microarray, using one ormore plastic reaction tube(s), using one or more glass vial(s), using achromatographic column, using a syringe, using a needle or using acombination of two or more thereof. It will be understood that thesurface of the used device should not or nearly not interfere with themethod, thus, in particular with the propensity for calcification.

In a preferred embodiment, the method is performed in one of thefollowing:

-   (a) a multiwell format, more preferably in an 8-well chamber plate,    in a 16-well chamber plate, in a 96-well microtiterplate or in a    384-well microtiterplate, in particular in a 96-well    microtiterplate;-   (b) a flow-through cell; or-   (c) a microfluidic device.

As used herein, the term “flow-through cell” may be understood in thebroadest sense as a device wherein the sample of the body fluid isconducted through whereat the propensity for calcification isdetermined. Herein, the flow may be a steady flow or may be interruptedfor measurement.

The data obtained from the method of the present invention may beanalyzed manually, semi-automatically or automatically.

In general, the method may be performed offline or online.

As used herein, the term “offline” may be understood in the broadestsense as any method wherein a body fluid sample is provided in a firststep (e.g., the patient's body, a preserved sample, or a sample of atechnical fluid) and steps (i) to (iii) of the method of the presentinvention are performed subsequently. Then, the method is performedbatchwise. Exemplarily, the propensity for calcification of a body fluidobtained from a dialysis patient may be diagnosed offline before, whileor after the patient's blood circulation is connected with the dialysismachine. The same may apply for a sample measured offline before used ina technical device (e.g., a cooling fluid used in a cooling circuit or alaundry agent in a washing machine).

As used herein, the term “online” may be understood in the broadestsense as any method wherein the provision of the body fluid sample andsteps (i) to (iii) of the method of the present invention are performedextensively simultaneously. Then, the body fluid is provided, conductedthrough a device and simultaneously measured. Herein, the flow may be asteady flow or may be interrupted for measurement. Optionally, the bodyfluid is extracted directly from a patient. Optionally, the analyzedbody fluid or a fraction thereof is conducted back into the patient'sbody. An online devise may optionally even be incorporated into thepatient's body. The same may apply for a sample measured online from atechnical device (e.g., a cooling circuit). Typically, an onlinemeasurement is a measurement over time. Exemplarily, the propensity forcalcification of a body fluid obtained from a dialysis patient may bediagnosed online when the patient's blood circulation is connected withthe dialysis machine. The method of the present invention may then beperformed automatically or semi-automatically. The dialysis machine mayeven comprise a device performing the method of the present invention asan intrinsic unit or as an add-on.

The measurement may be performed over time or may be a measurement atthe end point. Preferably, the measurement is a measurement over time.

As used in the context of the present invention, the term “over time”may be understood in the broadest sense as any measurement, whereinmeasurements are performed in one sample at different time points. Ameasurement over time may also be designated as a kinetic measurement.Exemplarily, the measurement over time may be kinetic nephelometry. Theperson skilled in the art will notice that the time interval chosenbetween two measurements will typically depend on the rate of theformation and/or rearrangement of particles. In order to determine fastformation and/or rearrangement of particles the person skilled in theart will typically chose short time intervals, whereas he will typicallychose longer time intervals for slower processes. The time intervals maytypically be in the range of few minutes up to several hours. Asmentioned above, the rate will typically depend on the insertedconcentrations and dilutions. Exemplarily, a measurement may beperformed over a total time of several seconds, several minutes, severalhours, several days or even several weeks. Preferably, in the context ofmeasurement over time, the measurement is started right after thecalcium and/or phosphate is added. As long as the reagent is constantthe rate of change can be seen as directly related to the amount of thepropensity of the body fluid for calcification. In order to also detectthe large particles, which may have settled to the bottom of the samplethe sample may optionally be shaken or mixed prior to each measurement.

As used herein, the term “end point” may be understood in the broadestsense as a measurement at a time point on which the formation orrearrangement is size of the CPPs is extensively completed and does notor nearly not change any more. In order to also detect the largeparticles, which may have settled to the bottom of the sample the samplemay optionally be shaken or mixed prior to measurement.

The method of the present invention may be performed by any principlesuitable for this method known in the art. The step of determining thetransition rate of the transition of primary CPPs into secondary CPPs(step (iii)) may be performed by any method known in the art. Preferablysuch method is based on detecting the average size or the average massof the particles present in the sample.

As used herein, the term “microfluidic device” may be understood in thebroadest sense as any miniaturized diagnostic tool. The microfluidicdevice may be any microfluidic device known in the art suitable in thecontext of the present invention. The microarray may also be understoodas a lab-on-a-chip. Typically, the microfluidic device will be a twodimensional array on a solid substrate such as, e.g., a glass slide, aceramic slide, a metal slide, a silicon thin-film cell, a plastic slideor a compact disc (CD) format. In order to enable the optical method ofthe present invention, at least a part of the microfluidic device maypreferably be permeable for the light used for the method of the presentinvention. The microfluidic device may comprise one or more microfluidicchannel(s) and/or chamber(s). These channels may also be used for mixingthe sample. In a microfluidic device one or both of the processes offormation of primary and/or secondary CPPs and the transition of primaryCPPs into secondary CPPs may be accelerated. The microfluidic device mayalso be a flow-through cell. A microfluidic device may also be adiagnostic medical dipstick, in particular in the context of urine as afluid.

The method may be performed manually, semi-automatized or automatized.

In a preferred embodiment, at least the step of determining one or moreof the rate of the formation of primary and/or secondary CPPs, theamount of primary and/or secondary CPPs and/or the rate of thetransition of primary CPPs into secondary CPPs (step (iii)) isautomated, more preferably at least the step of incubating said sampleat conditions allowing the formation of calciprotein particles (CPPs)and the step the determining the transition rate of the transition ofprimary CPPs into secondary CPPs (steps (ii) and (iii)) are automated,in particular all of the steps (i), (ii) and (iii) are automated.

As used in the context of the present invention, the terms “automated”and “automatized” may be understood interchangeably in the broadestsense as the use of any control system(s) and/or informationtechnology/technologies, in particular computer-assisted processe(s), toreduce the need for human work in the method. Exemplarily,automatization as used herein may comprise the use of automatic pipettesand/or computer-assisted readout methods. Optionally, automatization mayeven include computer-assisted experimental design and/orcomputer-assisted analysis of the obtained data. Graphical display ofthe obtained results may also be included.

In a preferred embodiment, the primary CPPs have an average diametersmaller than 100 nm and the secondary CPPs have an average diameter oflarger than 100 nm.

More preferably, the size of primary CPPs is in the range of between 50nm and 100 nm, most preferably in the range of about 60 nm to 75 nm. Thesecondary CPPs may more preferably have a size in the range of between100 nm and 500 nm, most typically in the range of about 100 nm to 200nm. However, as mentioned above it will be understood by a personskilled in the art that the size of the primary CPPs as well as the sizeof the secondary CPPs will typically depend on the specificconcentrations and conditions used in the method.

The transition rate of the transition of primary CPPs into secondaryCPPs determined for the body fluid may be analyzed by comparingdifferent body fluid samples with another or may be compared with one ormore calibration sample(s).

In a preferred embodiments, one or more of determining the rate of theformation of primary and/or secondary CPPs (a), determining the amountof primary and/or secondary CPPs (b) and/or determining the rate of thetransition of primary CPPs into secondary CPPs (c) step (iii) is/arecompared with one or more control sample(s).

As used in the context of the present invention, the term “controlsample” may be understood in the broadest sense as any sample that isusable as a reference sample. As used herein, the terms “control sample”and “calibration sample” may be understood interchangeably. The controlsample may be standardized sample (e.g., a sample with known properties)or may be an intrinsic control, i.e., one of the samples tested.Typically, under extensively identical reaction conditions (e.g.,measured at extensively the same temperature, in extensively the samebuffer, at extensively the same pH, with extensively the sameconcentrations of soluble calcium salt and soluble phosphate salt) andthe employment of an extensively identical method, the control samplealso will show extensively identical results. The propensity forcalcification of the sample(s) in question and the control sample(s) maybe measured concomitantly in assay (e.g., on one microtiter plate or onor in one microfluidic device), may be measured subsequently on the sameday (e.g., in a cuvette) or may be even measured on different days(e.g., the device used for determining the propensity of calcificationhas been calibrated by a control sample or is calibrated by a controlsample afterwards).

The use of a control sample may lead to the standardization of themeasurement and, thus, renders the method device-independent as theobtained results are compared with and referred to this control sampleon the device the measurement is also conducted on. Changes may then bequantified as changes relative to the control sample. The calibrationssample(s) may comprise one or more body fluid(s) or may be an artificialsample. Optionally, an artificial sample may bear a definedconcentration of inhibitors of the transition of primary CPPs intosecondary CPPs such as, e.g., a fetuin polypeptide (e.g., fetuin-A) oran albumin polypeptide (e.g., serum albumin).

When the fluid is a body fluid, the control sample in the context of thepresent invention may preferably be a sample obtained from a healthypatient. When the fluid is a technical fluid, an artificial body fluidor an infusion solution, the control sample in the context of thepresent invention may preferably be a sample bearing acceptablecalcification propensity. In case the propensity for calcification isconsiderably higher than that obtained for the control sample, this mayindicate that the propensity for calcification is undesirably high andthat further actions (e.g., the administration or use of calcificationinhibitors) may optionally be desired. In case the propensity forcalcification is considerably lower than that obtained for the controlsample, this may indicate that the propensity for calcification isundesirably low and that further actions (e.g., the administration oruse of calcification promoters, calcium and/or phosphate) may optionallybe desired.

The data obtained from any method(s) of the present invention may beanalyzed by any measure known in the art.

In a preferred embodiment, determining the rate of the transition ofprimary CPPs into secondary CPPs (c) of step (iii) is determined bydetermining the time point of half maximal transition time (T₅₀) of thetransition of primary CPPs into secondary CPPs.

As used in the context of the present invention, the term “half maximaltransition time” or “T₅₀” may be understood in the broadest sense as thetime point wherein half of the transition (50% transition) of primaryCPPs into secondary CPPs is accomplished. The dimension of the T₅₀ istime, thus, given in the unit second (s or sec), minute (min), hour (h),day (d), week (w), month or year (a). Most typically, the T₅₀ will begiven in minutes.

The T₅₀ value may be determined by plotting the measured lightscattering of the sample (e.g., in Relative Nephelometric Units (RNU))against the time (e.g., in [min]), exemplarily by plotting an XY-graph(cf, FIG. 1B.). The plotted data may be fitted by a nonlinear regression(e.g., in a log(agonist) vs. response—variable slope). Then, the lightscattering in the sample in which only primary CPPs are present isdetermined as well as the light scattering in the sample in which onlysecondary CPPs are present. The arithmetic mean between lightscatterings is calculated. Finally, the time point (T₅₀) in which theoverall light scattering represents such arithmetic mean is determined.In this context, the T₅₀ value may also be designated as RNU_(T50)value.

The method of the present invention may optionally further comprise thecalculation of the hydrodynamic radius (Rh) from the second ordercumulant fits via the Stokes-Einstein equation well-known in the art.Then, the T₅₀ value may also be determined by plotting the determinedaverage diameter sizes of the CPPs in the sample (e.g., in [nm]) againstthe time (e.g., in [min]), exemplarily by plotting an XY-graph (cf, FIG.1A). The plotted data may be fitted by a nonlinear regression (e.g., ina log(agonist) vs. response—variable slope). Then, the average particlesize of primary CPPs and the average particle size of secondary CPPs aredetermined. The arithmetic mean between the average particle size ofprimary CPPs and the average particle size of secondary CPPs iscalculated. Finally, the time point (T₅₀) in which the average particlesize of all CPPs represents such arithmetic mean is determined.

Plotting the data, fitting of the data and/or calculating the T₅₀ (orRNU_(T50)) value(s), may be performed manually or may be preformed by acomputer-assisted method. The computer-assisted method may be performedby any suitable program, e.g., by Excel® and/or GraphPad Prism®.

The method of the present invention may be used for scientific,industrial and/or clinical purposes. Exemplarily, it may be used toobtain more information on the interaction between congenital and/oracquired predisposition of the propensity for calcification in apatient. Further, the method may also be used to identify andcharacterize calcification inhibitors and promoters. The method may evenbe used for the primary and/or secondary screening of compounds in orderto determine their influence on calcification. This screening may be acell-free screening and optionally a high-throughput screening.

In the context of identifying and characterizing calcificationinhibitors and promoters, an inhibitor or promoter may be administeredto a patient or may be admixed to the body fluid in vitro. When theinhibitor or promoter may be administered to a patient, then, after acertain time, a sample from said patient is obtained and tested by themethod of the present invention. When inhibitor or promoter is admixedto the body fluid in vitro, then the sample is incubated for a certaintime after admixing the inhibitor or promoter and, subsequently, themethod of the present invention is conducted.

The method of the present invention may serve to identify unknowncalcification inhibitors and promoters and to test already knowncharacterize calcification inhibitors and promoters in more detail.Further, the interplay between different one or more inhibitor(s) and/orone or more promoter(s) may be investigated. The inhibitor(s) andpromoter(s) may be compound(s) active in a patient or in a medicalproduct or composition (e.g., a blood preservation, at the surface of amedical device). Alternatively, the inhibitor(s) and promoter(s) mayalso be compound(s) active in a non-medical context. Exemplarily,inhibitor(s) of calcification may be used in consumer goods (e.g.,laundry agents, dishwashing agents, domestic cleaning agents, toiletcleaning agents, aquarium cleaning agents etc.) and water additive(s).

Moreover, the method of the present invention may also be used todetermine the risk of a patient for developing calcification. Then, thedoctor and/or medicinal staff may obtain more information on whether andhow the patient should be treated with calcification inhibitors, thus,is able to make informed therapy decisions.

The following figures and examples are intended to illustrate theinvention but not to limit the scope of protection conferred by theclaims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1D. Detection of CPP transition. (FIG. 1A) 3D-DLS detection ofCPP transition in the presence of fetuin-A and human serum. (FIG. 2B)Nephelometry detection of CPP transition in the presence of human serum.(FIG. 1C and FIG. 1D) Gross visual appearance of the standardnephelometer assay serum solutions containing CPPs in solution (FIG. 1C)and after sharp centrifugation of the solutions (FIG. 1D). Experimentwas performed at 37° C. in standard photometry vials and with the samesolutions used in the respective proportions as in the finalnephelometer assay.

FIG. 2A-2D. Determination of nephelometer assay conditions. (FIG. 2A)Impact of varying calcium and phosphate concentrations. Calcium 10 mMand phosphate 6 mM were finally chosen as standard concentrations forthe assay. (FIG. 2B) Temperature-dependence of pH. Calcium or phosphatesolutions buffered by Hepes or Tris were heated from room temperature to40° C. and the pH values recorded. The less temperature sensitive Hepesbuffer was chosen for the assay. (FIG. 2C) Impact of pH. Calcium andphosphate solutions adjusted to pH values from 7.1 to 7.6 were tested. ApH of 7.40 at 37° C. was chosen for the assay. (FIG. 2D) Impact ofamount of serum. Serum amounts from 60 μl to 100 μl were tested (withNaCl 140 mM replenishing the missing volume to an amount 200 μl in allvials), and 80 μl serum finally chosen for the assay.

FIG. 3A-3B. Nephelometer assay conditions. (FIG. 3A) Assay results whenperformed with pooled serum from healthy volunteers with theNephelostar® instrument run at room temperature and the internalradiator set at 37° C. (FIG. 3B) Assay results when performed withpooled serum from healthy volunteers with the Nephelostar® radiatorturned off and the assay run in a temperature controlled room set at34.5° C. The resulting measurement temperature within the Nephelostar®plate holder bay under these conditions was 36.5 to 37° C.

FIG. 4A-4F. Nephelometer assay in calcifying animal models. (FIG. 4A)Illustrating representative x-rays of 10 to 16 week old dba2 fetuin-Aknock-out (−/−) and wildtype (+/+) mice showing excessive pathologicalcalcifications of fetuin-A knock-out mice. Heterozygous mice have thesame phenotype as wildtype mice. (FIG. 4B) Discrimination of wild type(wt), heterozygous (het) and knock out (ko) mouse sera. (FIG. 4C)Results of nephelometer assay performed with serum from fetuin-Aknock-out, heterozygous and wildtype mice. (FIG. 4D) Illustratingrepresentative histology of aortas from 16 week old adenine-treateduremic rats with calcifications of the vessel media (Alizarin stain forcalcium), and healthy rats without calcifications. (FIG. 4E) Comparisonbetween samples from uremic and non-uremic animals. (FIG. 4F)Nephelometry assay with sera from 20 hemodialysis patients (black) and20 healthy volunteers (grey).

FIG. 5A-5D. Particle characterization, microscopic appearance andmolecular composition of the CPPs. (FIG. 5A) Scanning electronmicroscopy (SEM) (left) and transmission electron microscopy (TEM)(right) of primary CPPs. (FIG. 5B) SEM (left) and TEM (right) ofsecondary CPPs. (FIG. 5C) Coomassie blue stain of protein contents ofprimary and secondary CPPs (left) and albumin and fetuin-A western blotsof primary and secondary CPPs (right). (FIG. 5D) Disappearance ofphosphate from the solution upon formation of CPPs.

FIG. 6A-6C. Assay dependence of spiked serum components. (FIG. 6A)Nephelometry in the absence of serum: only spiking of fetuin A, thestrongest intrinsic serum calcification inhibitor significantlyinfluences the assay. (FIG. 6B) Nephelometry in the presence of serum:all spiked substances influence the assay. (FIG. 6C) Alternativepresentation of the data shown in FIG. 6B. Substance concentrations usedin (FIG. 6A) and (FIG. 6B) were the same as given in the legend of (FIG.6C).

FIG. 7A-7B. Correlation of one-halfmaximal relative nephelometric units(RNU₅₀) and T₅₀ with fetuin-A serum concentrations. Fetuin-Aconcentrations were measured in sera obtained from 20 hemodialysispatients and plotted against the RNU₅₀ and T₅₀ values obtained from theassay of the present invention. Fetuin-A concentrations highlycorrelated with (FIG. 7A) RNU₅₀ (p=0.0006) and (FIG. 7B) T₅₀ (p=0.0413).Patient sera were the same as used for the experiment shown in FIG. 4F.Fetuin-A serum concentrations were measured by ELISA as described byKetteler M, et al. (Kettler et al., 2003).

EXAMPLES Methods Sampling and Preparation of Serum Specimens

Venous blood from eight healthy volunteers was taken in SarstedtMonovette vials. After clotting for 30 min, the samples were centrifugedat 3,000×g for 10 minutes at room temperature. Serum from allindividuals was pooled and aliquoted. Blood from 10 to 16 week old DBA/2fetuin-A knock-out, heterozygous and wildtype mice (Schafer et al.,2003; Jahnen-Dechent et al., 1997), was sampled from the heart at thetime of sacrifice.

Blood from male Wistar rats (Charles River, Sulzfeld, Germany), whichhad received food supplemented with 0.75% adenine and calcium 1.05%,phosphorus 0.8%, protein 18.5% for four weeks to induce uremia andvascular calcifications, was taken at sacrifice at age 16 weeks from theinferior vena cava (Pasch et al., 2008). Likewise, control blood wastaken from healthy, nonuremic, non-calcified rats of the same age andgender, which had been treated with sodium thiosulfate (0.4 g/kg bodyweight) in normal (0.9%) saline i.p. three times a week for 6 weeks. Ofnote, sodium thiosulfate (Na₂5₂O₃) did not have any impact on the assaywhen spiked to serum in amounts of up to 40 mM.

After clotting at room temperature, blood samples from humans, mice orrats were spun at 3,000×g for 10 minutes at room temperature to separateserum from blood cells. The serum was shock frozen in liquid nitrogenand stored at −80° C. Before use in the nephelometer assay, samples werethawed, and centrifuged at 10,000×g for 30 min at room temperature toremove potential small particles which might have formed during thefreezing and thawing of samples (cryoprecipitates) and which mightinterfere with the assay by providing precipitation-acceleratingniduses.

Devices, Plastic Materials and Chemicals

The Nephelostar® nephelometer was purchased from bmg labtech, Offenburg,Germany, the Liquidator96™ bench-top pipetting system was purchased fromMettler Toledo GmbH, Giessen, Germany. 96-well plates were from BrandGmbH, Wertheim, Germany, and 96-well plasic Covers from Carl Roth GmbH,Karlsruhe, Germany. All chemicals (e.g., NaCl, Hepes, CaCl₂, NaH₂PO₄,Na₂HPO₄ and NaOH) were purchased from AppliChem, Darmstadt, Germany, in“pro analysi” grade quality.

Protein Quantification

For quantification of proteins in solutions, the Pierce BCA ProteinAssay Kit was used according to the manufacturer's instructions. BSA (2mg/ml, Pierce) was used as a standard. Western blots were performedaccording to standard protocols with SDS-PAGE (4%-12%), with 1 mgprotein or 0.4 mg pure fetuin-A or albumin loaded per lane. Thefollowing primary antibodies against fetuin-A and albumin were used:polyclonal rabbit anti-human fetuin-A antiserum 5359 (Behring AG,Marburg, Germany) and mouse anti-human albumin (1:2500, catalog number0300-0080; AbD Serotec). For fluorescence detection, the followinghorseradish peroxidase-coupled secondary antibodies were used: swineanti-rabbit IgG (1:5000, catalog number P0217; Dako) and rabbitanti-mouse IgG (1:2000, catalog number P0260; Dako). Protein stains wereperformed with the Imperial Protein Stain according to themanufacturer's instructions (Thermo Scientific); 6.0 mg total protein or2.5 mg pure fetuin-A or albumin was loaded per lane.

Three-Dimensional Cross-Correlation Dynamic Light Scattering (3D-DLS)

Multiple scattering in Solutions with high particle density prevents thecharacterization by Standard dynamic lightscattering methods. Thereforewe used a 3D cross-correlation dynamic light scattering (3D-DLS) setupfor the analysis of turbid CPP samples. 50-52 Measurements wereperformed using a Standard light scattering device (ALV GmbH, Langen,Germany) with He—Ne-laser (JDS Uniphase, Koheras GmbH, 632.8 nm, 25 mW,Type LGTC 685-35), two avalanche photodiodes (Perkin Eimer, TypeSPCM-AQR-13-FC) and an ALV 5000 correlator. The scattered light wasdetected at 90° geometry. The sample temperature was adjusted by anexternal thermostat equipped with a Pt-100 temperature sensor. Thehydrodynamic radius Rh was calculated from second-order cumulant fitsvia the Stokes-Einstein equation. Measurements covered a time span of1400 minutes in 2 minute intervals. Previous TEM investigations revealedthat aged, secondary CPPs have an ellipsoidal shape with an axes ratioof approximately b/a z 0.3. For the sake of clarity, we calculated thehydrodynamic radii, not the semi-axes, to characterize the individualCPP stages.

Nephelometer Assay

Three-dimensional cross-correlation dynamic light scattering (3D-DLS) isa method, which detects laser scatter in solutions and integrates thesedata to yield Information about the development of particle size overtime.

Stock Solutions: 1. NaCl-solution: NaCl 140 mM, 2. Calcium solution:CaCl₂ 40 mM+Hepes 100 mM+NaCl 140 mM, pH adjusted with NaOH 10 mM to7.40 at 37° C., 3. Phosphate solution: Na₂HPO₄ 19.44 mM+NaH₂PO₄ 4.56mM+Hepes 100 mM+NaCl 140 mM, pH adjusted with NaOH 10 mM to 7.40 at 37°C. Preparation of 96-well plates: all Solutions were pre-warmed to 34.5°C. in a thermo constant room where also all pipetting steps wereperformed with the liquidator96™ bench-top pipetting System using a setof new pipetting tips for every pipetting step. These pipetting stepswere performed in the following order: 1. NaCl-solution: 20 μl/well, 2.serum 80 μl/well, 3. shaking for 1 minute, 4. phosphate solution 50μl/well, 5. shaking for 1 minute, 6. calcium solution 50 μl/well,shaking for 1 minute. Air bubbles in the wells were disintegrated with apocket lighter and the 96-well covered with a ThinSeal™ adhesive sealingfilm for microplates. As line “A” of the 96-well plate often showedunreliable results, it was generally left out. Assay conditions andNephelostar® settings: measurement in a thermo constant room at 34.5° C.with the internal rediation of the device turned off. This led to aninfernal measurement temperature of 36.5° C. to 37° C. The Nephelostar®was operated and controlled via the Nephelostar provider's GalaxySoftware on a Windows Computer platform. The assay was performed with200 cycles of 1.5 seconds measurement time per well and a position delayof 0.1 seconds in horizontal plate reading mode, adding up to a cycletime of 180 seconds/cycle for our Standard assay. This adds up to atotal assay run time of 10 hours per assay. For some measurements, thecycle time was extended to 360 or 540 seconds, which adds up to assaytimes of 20 and 30 hours, respectively. The gain and laser adjustmentwas set at 90% required value, gain 50 with a laser beam focus of 1.5 mmand a laser intensity of 50%.

Data Processing

After completion of the run, data were transferred to Excel® andtransposed from lines into columns. Data columns were copied into theGraphPad Prism® program to generate an XY-graph. Data were thenprocessed by calculating nonlinear regression in the “log(agonist) vs.response—variable slope (four Parameters)” mode using the “robust fit”fitting method. The resulting values obtained for T₅₀ and RNU_(T50) werefurther processed as required.

Results

Here, we tested whether primary CPPs would also be generated when humanserum instead of fetuin-A solution was used (FIG. 1A, lower part).Indeed, in both cases primary CPPs of comparable size (diameter about 50nm) were generated, which underwent spontaneous transition to secondaryCPPs (diameter about 150 nm), albeit within very different time frames(FIG. 1A). Given these grossly different transition times, we reasonedthat the delay of the transition might reflect the stability of primaryCPPs and that measuring this step might provide a quantitative estimatefor the calcification inhibitory propensity inherent in serum.

As 3D-DLS is not widely available and can measure only one sample perday, we aimed to establish a practical and broadly applicablealternative assay for the detection of the mentioned transition step.Nephelometry is based on the same principles as DLS and quantifies theamount of laser light scatter in turbid solutions. Consequently, thetransition was also detectable by nephelometry (FIG. 1B), and it is ofnote even visible to the naked eye (FIG. 1C).

For the establishment of the nephelometry-based assay, we took advantageof the automated laser-based microplate nephelometer Nephelostar® whichwe run in the 96-well-plate mode. The resulting data were analyzed usingExcel® and GraphPad Prism® Software to yield nonlinear regression curvesand the resulting values of half maximal transition time (T₅₀, FIG. 1A))and of Relative Nephelometric Units (RNU_(T50), FIG. 1B) at this pointin time were determined.

We chose physiological conditions for temperature (37° C.) and pH (7.40at 37° C.), and designed the assay for a final volume of 200 μl perwell. These 200 μl consisted of 20 μl NaCl 140 mM, 80 μl serum, 50 μlphosphate 24 mM and 50 μl calcium 40 mM solution which were mixed inthis order. The phosphate and calcium Solutions were supplemented withNaCl 140 mM and Hepes 100 mM, the pH was adjusted to 7.40 at 37° C. Thismixture resulted in the final concentrations as depicted below:

-   Ca²⁺: 10 mM-   PO₄ ²⁻: 6 mM-   NaCl: 140 mM-   Hepes: 50 mM-   at a pH 7.40 and 37° C.

The 20 μl NaCl were introduced as an extra volume usable for spikingexperiments. A wide range of calcium and phosphate concentrations weresystematically tested (FIG. 2A) and final concentrations of calcium 10mM (i.e. 40 mM in stock solution) and phosphate 6 mM (i.e. 24 mM instock solution) finally chosen in our assay for three reasons: (i) thetransition occurred in a central position in the time- and RNUcoordinate system leaving space for changes into all directions, (ii)these concentrations represent the numerically optimal relation ofcalcium and phosphate with regard to the formation of hydroxylapatite,(iii) our previous experiments investigating CPPs have been performedwith these concentrations.

Unfortunately, first attempts to standardize the assay showed anenormous variation even within a given 96-well plate (FIG. 3A),indicating that the transition step is an extremely sensitivephysicochemical process, which is sensitive towards subtle variations inpH (FIG. 2C) and serum amounts used (FIG. 2D).

Stabilization of the assay (FIG. 3B) was achieved by introducing threeimportant modifications: (i) assay temperature was stabilized by runningthe assay in a Special constant temperature room with the intrinsicradiator of the Nephelostar® turned off, (ii) pipetting volumes werestabilized by using a high precision 96-well pipetting device (theLiquidator96™) instead of multi-channel pipettes, and (iii)temperature-sensitivity of the test was diminished by using Hepesinstead of the more temperature-sensitive Tris buffer (FIG. 2B). Underthese conditions, the assay was stable and yielded highly reproducibleresults (FIG. 3B) with an intra-day variability of +/−5.2% and aninter-day variability of +/−11.6% in our hands.

To confirm the correlation between the assay results and calcificationsin vivo, we compared serum of fetuin-A knock-out (ko), heterozygous(het) and wildtype (wt) mice (FIGS. 4A, B and C) and found that T₅₀ wasshorter in serum from the knockout mouse (ko), when compared toheterozygous and wildtype mice. The same pattern was found when serumfrom calcifying uremic rats and from healthy non-calcifying rats wascompared (FIGS. 4D and E). Here again the transition (T₅₀) occurredearlier in the calcifying than in the non-calcifying animals.

We also tested sera from healthy volunteers and hemodialysis patients.Again, the test discriminated the calcification-prone from thenoncalcificationprone individuals (i.e., the hemodialysis patients fromthe healthy volunteers (FIG. 4F), indicating that the test reflectscalcification propensity in serum.

These results confirm that the nephelometer assay presented hereprovides an estimate of intrinsic serum-related calcificationpropensity.

The test of the present invention increases supersaturation of serum byadding Ca (10 mM) and phosphate (6 mM). The specific effect ofsupersaturation depends on the intrinsic concentrations of fetuin-A,albumin, phosphate etc. in a given serum. As a general rule, the higherthe calcium and phosphate supersaturation, the lower T₅₀ and the higherRNU₅₀. This applies to sera from HD patients and healthy volunteersalike. RNU₅₀ largely depends on the protein content (fetuin-A, albumin)of the CPPs with some contribution of phosphate. T₅₀ largely depends onMg and phosphate with some contribution of fetuin-A and albumin. A lowT₅₀ is therefore often associated with a high RNU₅₀ and vice versa. Auniversal RNU₅₀-to-T₅₀-ratio can however, not be determined as bothvariables depend on different—albeit overlapping—determinants.

In summary, we present a nephelometer-based assay, which measurescalcification propensity of a body fluid, exemplarily shown in bloodserum. Given the wide area of potential applications of this assay, thismethod is a useful tool for the investigation and elucidation ofbiomineralization-related issues in clinical as well as scientificresearch and in diagnosis in vivo and ex vivo.

REFERENCES

-   1. Heiss, A. et al. (2003), Structural basis of calcification    inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of    colloidal CPPs, J Biol Chem 278, 13333-13341.-   2. Heiss, A. et al. (2008), Hierarchical role of fetuin-A and acidic    serum proteins in the formation and stabilization of calcium    phosphate particles, J Biol Chem 283, 14815-14825.-   3. Jahnen-Dechent, W. et al. (1997), Cloning and targeted deletion    of the mouse fetuin gene, J Biol Chem 272, 31496-31503.-   4. Jahnen-Dechent, W., Schafer, C, Heiss, A. and Grotzinger, J.    (2001), Systemic inhibition of spontaneous calcification by the    serum protein alpha 2-HS glycoprotein/fetuin, Z Kardiol 90 Suppl    3,47-5.-   5. Jahnen-Dechent, W., Heiss, A., Schafer, C., Kettler, M. (2011),    Fetuin-A Regulation of Calcified Matrix Metabolism, Circulation    Research 108, 1494-1509.-   6. Ketteler M, Bongartz P, Westenfeld R, Wildberger J E, Mahnken A    H, Bohm R, Metzger T, Wanner C, Jahnen-Dechent W, Floege J (2003),    Association of low fetuin-A (AHSG) concentrations in serum with    cardiovascular mortality in patients on dialysis: a cross-sectional    study, Lancet 361: 827-833.-   7. Pasch, A. et al. (2008), Sodium thiosulfate prevents vascular    calcifications in uremic rats, Kidney Int 74, 1444-1453.-   8. Reynolds, J. L., et al. (2005), Multifunctional roles for serum    protein fetuin-a in Inhibition of human vascular smooth muscle cell    calcification, J Am Soc Nephrol 16, 2920-2930-   9. Reynolds, J. L., et al. (2004), Human vascular smooth muscle    cells undergo vesicle-mediated calcification in response to changes    in extracellular calcium and phosphate concentrations: a potential    mechanism for accelerated vascular calcification in ESRD, J Am Soc    Nephrol 15, 2857-2867.-   10. Schäfer, C. et al. (2003), The serum protein alpha    2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting    inhibitor of ectopic calcification, J Clin Invest 112, 357-366.-   11. Wald, J., et al. (2011), Formation and stability kinetics of    calcium phosphate-fetuin-A colloidal particles probed by    time-resolved dynamic light scattering, Soft Matter.-   12. Wu, C. Y., Martel, J., Young, D. and Young, J. D. (2009),    Fetuin-A/albumin-mineral complexes resembling serum calcium granules    and putative nanobacteria: demonstration of a dual    inhibition-seeding concept, PLoS One 4, e8058.-   13. Young, J. D., et al. (2009), Putative nanobacteria represent    physiological remnants and culture by-products of normal calcium    homeostasis, PLoS One 4, e4417.-   14. Young, J. D., et al. (2009), Characterization of granulations of    calcium and apatite in serum as pleomorphic mineralo-protein    complexes and as precursors of putative nanobacteria. PLoS One 4,    e5421.-   15. Yusuf, S., Reddy, S., Ounpuu, S. and Anand, S. (2001), Global    burden of cardiovascular diseases: part I: general considerations,    the epidemiologic transition, risk factors, and impact of    urbanization, Circulation 104, 2746-2753.

What is claimed is:
 1. A method for determining the propensity of a body fluid obtained from an individual for calcification comprising: (i) adding a soluble calcium salt and a soluble phosphate salt to a sample of said body fluid; (ii) incubating said sample at conditions allowing the formation of calciprotein particles (CPPs); and (iii) determining one or more of the following: (a) a rate of the formation of primary and/or secondary CPPs; (b) an amount of primary and/or secondary CPPs; and/or (c) a rate of the transition of primary CPPs into secondary CPPs, wherein an increase over what is determinable for a fluid with known calcification in one or more of (a), (b) and/or (c) of step (iii) indicates an increased propensity of said body fluid for calcification.
 2. The method of claim 1, wherein step (iii) is performed by an optical method.
 3. The method of claim 2, wherein excitation light used in the optical method is a laser beam.
 4. The method of claim 2, wherein the optical method is performed by detecting light scattering.
 5. The method of claim 1, wherein step (iii) is performed by any method selected from the group consisting of: sedimentation techniques, filtration analysis, size exclusion chromatography, granulometry, acoustic spectroscopy, or a combination of two or more thereof.
 6. The method of claim 1, wherein the fluid is a body fluid obtained from a patient that has developed calcification and/or is at risk of developing calcification.
 7. The method of claim 6, wherein the patient suffers from vascular, valvular and/or soft tissue calcification.
 8. The method of claim 1, wherein said method is performed at a constant temperature and/or at a constant pH.
 9. The method of claim 1, wherein said method is performed in one of the following: (a) a multiwell format; (b) a flow-through cell; or (c) a microfluidic device
 10. The method of claim 1, wherein at least step (iii) is automated, or wherein at least steps (ii) and (iii) are automated, or wherein all of the steps (i), (ii) and (iii) are automated.
 11. The method of claim 1, wherein the primary CPPs have an average diameter smaller than 100 nm and the secondary CPPs have an average diameter of larger than 100 nm.
 12. The method of claim 1, wherein one or more of (a), (b) and/or (c) of step (iii) is/are compared with one or more control sample(s).
 13. The method of claim 1, wherein (c) of step (iii) is determined by determining a time point of half maximal transition time (T₅₀) of the transition of primary CPPs into secondary CPP s.
 14. The method of claim 2, wherein the optical method is a method selected from the group consisting of: absorptiometry, detection of light scattering, correlation spectroscopy, or a combination of two or more thereof
 15. The method of claim 4, wherein the method of detecting light scattering is selected from the group consisting of: dynamic light scattering, cross-correlation dynamic light scattering, three-dimensional cross-correlation dynamic light scattering, or nephelometry.
 16. The method of claim 1, wherein the body fluid is blood, blood plasma, blood serum, lymph, or urine.
 17. The method of claim 6, wherein said patient is a dialysis patient.
 18. The method of claim 7, wherein said patient further suffers from a rheumatoid disease, a malignant disease and/or an infectious disease, or wherein the patient shows at least one of the syndromes selected from the group consisting of: renal dysfunction, hypertension, diabetes mellitus, dyslipidemia, a lack of adequate mineralization, and atherosclerosis.
 19. The method of claim 18, wherein the lack of adequate mineralization is due to osteoporosis, osteomalacia, or a combination thereof. 